U.S. patent number 9,062,285 [Application Number 14/011,434] was granted by the patent office on 2015-06-23 for compositions and methods for modulating ampa receptor-mediated excitotoxicity.
The grantee listed for this patent is CENTRE FOR ADDICTION AND MEDICAL HEALTH. Invention is credited to Fang Liu.
United States Patent |
9,062,285 |
Liu |
June 23, 2015 |
**Please see images for:
( Certificate of Correction ) ** |
Compositions and methods for modulating AMPA receptor-mediated
excitotoxicity
Abstract
The present invention provides AMPAR excitotoxicity mediating
polypeptides comprising the GAPDH(2-2-1-1) (I221-E250)amino acid
sequence (SEQ ID NO:2). Also disclosed are nucleotide sequences
encoding the polypeptides, methods of inhibiting GAPDH association
with the GluR2 subunit or p53. Methods of inhibiting AMPA receptor
mediated excitotoxicity using the polypeptides and nucleic acids
are also disclosed.
Inventors: |
Liu; Fang (Toronto,
CA) |
Applicant: |
Name |
City |
State |
Country |
Type |
CENTRE FOR ADDICTION AND MEDICAL HEALTH |
Toronto |
N/A |
CA |
|
|
Family
ID: |
46455743 |
Appl.
No.: |
14/011,434 |
Filed: |
August 27, 2013 |
Prior Publication Data
|
|
|
|
Document
Identifier |
Publication Date |
|
US 20130336951 A1 |
Dec 19, 2013 |
|
Related U.S. Patent Documents
|
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
Issue Date |
|
|
13343555 |
Jan 4, 2012 |
8536115 |
|
|
|
12310558 |
Feb 21, 2012 |
8119768 |
|
|
|
PCT/CA2007/001539 |
Aug 31, 2007 |
|
|
|
|
60841195 |
Aug 31, 2006 |
|
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K
38/177 (20130101); C12N 9/0008 (20130101); A61P
25/08 (20180101); A61P 25/00 (20180101) |
Current International
Class: |
C07K
14/705 (20060101); C12N 9/02 (20060101); A61K
38/17 (20060101) |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
Other References
Lind et al., Studies on the Mechanism of Oxidative Modification of
Human Glyceraldehyde-3-phosphate Dehydrogenase by Glutathione:
Catalysis by Glutaredoxin, Jun. 18, 1998, Biochemical and
Biophysical Research Communications 247(2):481-486. cited by
examiner .
Chen, et al, "Stargazin regulates synaptic targeting of AMPA
receptors by two distinct mechanisms", Nature, Dec. 2000, vol. 408,
pp. 936-943. cited by applicant .
Choi, et al., "Isolation of the heterogeneous nuclear
RNA-ribonucleoprotein complex (hnRNP): A unique supramolecular
assembly", Proc. Natl. Acad. Sci., Dec. 1, 1984, vol. 81, No. 23,
pp. 7471-7475. cited by applicant .
Dong, et al., "Characterization of the Glutamate
Receptor-Interacting Proteins GRIP1 and GRIP2", J. Neurosci., Aug.
15, 1999, vol. 19(16), pp. 6930-6941. cited by applicant .
Doucet, et al., "Identification of Low Molecular Weight GTP-binding
Proteins and Their Sites of Interaction in Subcellular Fractions
from Skeletal Muscle", The Journal of Biological Chemistry, Sep.
15, 1991, vol. 266, No. 26, pp. 17613-17620. cited by applicant
.
Fuchtbauer, et al., "Actin-severing activity copurifies with
phosphofructokinase", Proc. Natl. Acad. Sci., Dec. 15, 1986, vol.
83, No. 24, pp. 9502-9506. cited by applicant .
Geiger, et al., Relative Abundance of Subunit mRNAs Determines
Gating and Ca2+ Permeability of AMPA Receptors in Principal Neurons
and Interneurons in Rat CNS, Neuron, Jul. 1995, vol. 15, pp.
193-204. cited by applicant .
Glaser, et al., "Rapid Plasmenylethanolamine-Selective Fusion of
Membrane Bilayers Catalyzed by an Isoform of
glyceraldehyde-3-Phosphate Dehydrogenase: Discrimination between
Glycolytic and Fusogenic Roles of Individual Isoforms",
Biochemistry, 1995, vol. 34, No. 38, pp. 12193-12203. cited by
applicant .
Hara, et al., "Neuroprotection by pharmacologic blockage of GAPDH
death cascade", Proc. Natl. Acad. Sci., Mar. 7, 2006, vol. 103, No.
10, pp. 3887-3889. cited by applicant .
Huitorel, et al., "Bundling of microtubules by
glyceraldehyde-3-phosphate dehydrogenase and its modulation by
ATP", Eur. J. Biochem., 1985, vol. 150, pp. 265-269. cited by
applicant .
Humbert, et al., "Inositol1, 4, 5-Trisphosphate Receptor Is Located
to the Inner Nuclear Membrane Vindicating Regulation of Nuclear
Calcium Signaling by Inositol 1, 4, 5-Trisphosphate", The Journal
of Biological Chemistry, Jan. 5, 1996, vol. 271, No. 1, pp.
478-485. cited by applicant .
Iihara, et al., The Influence of Glutamate Receptor 2 Expression on
Excitotoxicity in GluR2 Null Mutant Mice, J. Neurosci., Apr. 1,
2001, vol. 21(7), pp. 2224-2239. cited by applicant .
Ikemoto, et al., "Glycolysis and Glutamate Accumulation into
Synaptic Vesicles", The Journal of Biological Chemistry, Feb. 21,
2003, vol. 278, No. 8, pp. 5929-5940. cited by applicant .
Jonas, et al., "Differences in Ca2+ Permeability of AMP A-type
Glutamate Receptor Channels in Neocortical Neurons Caused by
Differential GluR-B Subunit Expression", Neuron, Jun. 1994, vol.
12, pp. 1281-1289. cited by applicant .
Jong, et al., "Functional Metabotropic Glutamate Receptors on
Nuclei from Brain and Primary Cultured Striatal Neurons", The
Journal of Biological Chemistry, Aug. 26, 2005, vol. 280, No. 34,
pp. 30469-30480. cited by applicant .
Jong, et al., "Nuclear localization of functional metabotropic
glutamate receptor mGiu1 in HEK293 cells and cortical neurons: role
in nuclear calcium mobilization and development", J. Neurochem.,
2007, vol. 101, pp. 458-469. cited by applicant .
Lakkaraju, et al., "Neurons Are Protected from Excitotoxic Death by
p53 Antisense Oligonucleotides Delivered in Anionic Liposomes", The
Journal of Biological Chemistry, Aug. 24, 2001, vol. 276, No. 34,
pp. 32000-32007. cited by applicant .
Lee, et al., "Dual Regulation of NMDA Receptor Functions by Direct
Protein-Protein Interactions with the Dopamine D 1 Receptor", Cell,
Oct. 18, 2002, vol. 111, pp. 219-230. cited by applicant .
Lee, et al., "Ciathrin Adaptor AP2 and NSF Interact with
Overlapping Sites of GluR2 and Play Distinct Roles in AMPA Receptor
Trafficking and Hippocampal LTD", Neuron, Nov. 14, 2002, vol. 36,
pp. 661-674. cited by applicant .
Li, et al, "Down-regulation of GluR2 is associated with
Ca2+-dependent protease activities in kainate-induced apoptotic
cell death in cultured [correction of culturd] rat hippocampal
neruons", Neuroscience Letters, 2003, vol. 352, pp. 105-108. cited
by applicant .
Lin, et al, "Distinct molecular mechanisms and divergent
endocytotic pathways of AMPA receptor internalization", 0 Nature
Neuroscience, Dec. 2000, vol. 3, No. 12, pp. 1282-1290. cited by
applicant .
Lin, et al., "Nuclear localization of EGF receptor and its
potential new role as a transcription factor", Nature Cell 0
Biology, Sep. 2001, vol. 3, pp. 802-808. cited by applicant .
Liu, et al., "Ischemic Insults Direct Glutamate Receptor Subunit
2-Lacking AMPA Receptors to Synaptic Sites", J. Neurosci., May 17,
2006, vol. 26(20), pp. 5309-5319. cited by applicant .
Liu, et al., "Direct protein-protein coupling enables cross-talk
between dopamine D5 and gamma-aminobutyric acid A receptors",
Nature, Jan. 20, 2000, vol. 403, pp. 274-280. cited by applicant
.
Liu et al., "Expression of Ca2+-Permeable AMPA Receptor Channels
Primes Cell Death in Transient Forebrain Ischemia", Neuron, Jul. 8,
2004, vol. 43, pp. 43-55. cited by applicant .
Lu, et al., "Angiotensin 11-Induced Nuclear Tarageting of the
Angiotensin Type 1 (AT1) Receptor in Brain Neurons", Endocrinology,
1998, vol. 139, No. 1, pp. 365-375. cited by applicant .
Man, et al., "Regulation of AMPA Receptor-Mediated Synaptic
Transmission by Clathrin-Dependent Receptor Internalization",
Neuron., Mar. 2000, vol. 25, pp. 649-662. cited by applicant .
Melikian, et al, "Membrane Trafficking Regulates the Activity of
the Human Dopamine Transporter", J. Neurosci., Sep. 15, 1999, vol.
19(18), pp. 7699-7710. cited by applicant .
Miller, et al., "Neuronal life and death: an essential role for the
p53 family", Cell Death and Differentiation, 2000, vol. 7, pp.
880-888. cited by applicant .
Nelson, et al., "pH-regulated Secretion of a
Glyceraldehyde-3-Phosphate Dehydrogenase from Streptococcus
gordonii FSS2: Purification, Characterization and Cloning of the
Gene Encoding this Enzyme", Journal of Dental Research, Jan. 2001,
vol. 80(1), pp. 371-377. cited by applicant .
Nishimune, et al, "NSF Binding to GluR2 Regulates Synaptic
Transmission", Neuron., Jul. 1998, vol. 21, pp. 87-97. cited by
applicant .
Robbins, et al., "A Mutation in Glyceraldehyde 3-Phosphate
Dehydrogenase Alters Endocytosis in CHO Cells", The Journal of Cell
Biology, Sep. 1, 1995, vol. 130, pp. 1093-1104. cited by applicant
.
Saglietti, et al, Extracellular Interactions between GluR2 and
N-Cadherin in Spine Regulation, Neuron, May 3, 2007, vol. 54, pp.
461-477. cited by applicant .
Sakhi, et al., "p53 induction is associated with neuronal damage in
the central nervous system", Proc. Natl. Acad. Sci., Aug. 2, 1994,
vol. 91, No. 16, pp. 7525-7529. cited by applicant .
Seifert, et al., "Characterization of group 8 streptococcal
glyceraldehyde-3-phosphate dehydrogenase: surface localization,
enzymatic activity, and protein-protein interactions", Can. J.
Microbial., 2003, vol. 49, pp. 350-356. cited by applicant .
Srivastava, et al., "Novel Anchorage of GluR2/3 to the Postsynaptic
Density by the AMPA Receptor-Binding Protein ABP", Neuron, Sep.
1998, vol. 21, pp. 581-591. cited by applicant .
Uberti, et al., "Induction of tumour-suppressor phosphoprotein p53
in the apoptosis of cultured rat cerebellar neurones triggered by
excitatory amino acids", European Journal of Neuroscience, 1998,
vol. 10, pp. 246-254. cited by applicant .
Valtschanoff, et al., "SAP97 concentrates at the postsynaptic
density in cerebral cortex", European Journal of Neuroscience,
2000, vol. 12, pp. 3605-3614. cited by applicant .
Ventura, et al., "Nuclear Opioid Receptors Activate Opioid Peptide
Gene Transcription in Isolated Myocardial Nuclei", The Journal of
Biological Chemistry, May 29, 1998, vol. 273, No. 22, pp.
13383-13386. cited by applicant .
Wyszynski, et al., "Associate of AMPA Receptors with a Subset of
Glutamate Receptor-Interacting Protein in Vivo", J. Neurosci., Aug.
1, 1999, vol. 19(15), pp. 6528-6537. cited by applicant .
Xia, et al, "Clustering of AMPA Receptors by the Synaptic PDZ
Domain-Containing Protein PICK1", Neuron. Jan. 1999, vol. 22, pp.
179-187. cited by applicant .
Yamaji, et al., "Giyceraldehyde-3-phosphate dehydrogenase in the
extracellular space inhibits cell spreading", Biochimica et
Biophysics Acta, 2005, vol. 1726, pp. 261-271. cited by applicant
.
Zeevalk, et al., "Excitotoxicity at Both NMDA and Non-NMDA
Glutamate Receptors Is Antagonized by Aurintricarboxylic Acid:
Evidence for Differing Mechanisms of Action", J. Neurochem., 1995,
vol. 64, No. 4, pp. 1749-1758. cited by applicant .
Beretta, et al., "NSF interaction is important for direct insertion
of GluR2 at synaptic sites", Mol. Cell. Neurosci., 2005, vol. 28,
pp. 650-660. cited by applicant .
Passafaro et al., "Induction of dendritic spines by an
extracellular domain of AMPA receptor subunit GluR2," Nature,
424:677-681 (Aug. 7, 2003). cited by applicant .
Chuang, D-M et al., "Gyceraldehyde-3-phosphate dehydrogenase,
apoptosis and neurodegenerative diseases", Ann. Rev. Pharm. and
Tox., Feb. 7, 2005, vol. 45, pp. 269-290, ISSN: 0362-1642. cited by
applicant .
Hollmann, et al., "Cloned Glutamate Receptors", Annu. Rev.
Neurosci., 1994, vol. 17, pp. 31-108. cited by applicant .
Bliss, et al., "A synaptic model of memory: long-term potentiation
in the hippocampus", Nature, Jan. 7, 1993, vol. 361, pp. 31-39.
cited by applicant .
Simon, et al., "Blockade of N-Methyi-0-Aspartate Receptors May
Protect Against Ischemic Damage in the Brain", Science, Nov. 16,
1984, vol. 226, pp. 850-852. cited by applicant .
Choi, "Calcium: still center-stage in hypoxic-ischemic neuronal
death", Trends Neurosci., 1995, vol. 18, No. 2, pp. 58-60. cited by
applicant .
Lee, et al., "The changing landscape of ischaemic brain injury
mechanisms", Nature, Jun. 24, 1999, vol. 399/Supp, pp. A7-A14.
cited by applicant .
Pulsinelli, et al., "Regional Cerebral Blood Flow and Glucose
Metabolism Following Transient Forebrain Ischemia", Annals of
Neurology, May 1982, vol. 11, No. 5, pp. 499-509. cited by
applicant .
Schmidt-Kastner, et al., "Selective Vulnerability of the
Hippocampus in Brain Ischemia", Neuroscience, 1991, vol. 40, No. 3,
pp. 599-636. cited by applicant .
Pellegrini-Giampietro, et al., "Switch in glutamate receptor
subunit gene expression in CA1 subfield of hippocampus following
global ischemia in rats", Proc. Natl. Acad. Sci., Nov. 1992, vol.
89, pp. 10499-10503. cited by applicant .
Gill, et al., "Pharmacology of AMPA Antagonists and Their Role in
Neuroprotection", Int. Rev. Neurobiol., 1997, vol. 40, pp. 197-232.
cited by applicant .
Oguro, et al., "Knockdown of AMPA Receptor GluR2 Expression Causes
Delayed Neurodegeneration and Increases Damage by Sublethal
Ischemia in Hippocampal CA1 and CA3 Neurons", J. Neurosci., Nov. 1,
1999, vol. 19(21), pp. 9218-9227. cited by applicant .
Weiss, et al., "Ca2+-Zn2+ permeable AMPA or kainate receptors:
possible key factors in selective neurodegeneration", Trends
Neurosci., 2000, vol. 23, No. 8, pp. 365-371. cited by applicant
.
Yin, et al., "Blockage of Ca2+--Permeable AMPA/Kainate Channels
Decreases Oxygen-Glucose Deprivation-Induced Zn2+ Accumulation and
Neuronal loss in Hippocampal Pyramidal Neurons", J. Neurosci., Feb.
15, 2002, vol. 22(4), pp. 1273-1279. cited by applicant .
Wang, et al., "Cdk5 activation induces hippocampal CA 1 cell death
by directly phosphorylating NMDA receptors", Nature Neuroscience,
Oct. 2003, vol. 6, No. 10, pp. 1039-1047. cited by applicant .
Greengard, et al., "Enhancement of the Glultamate Response by
cAMP-Dependent Protein Kinase in Hippocampal Neurons", Science,
Sep. 6, 1991, vol. 253, No. 5024, pp. 1135-1138. cited by applicant
.
Wang, et al., "Modulation of AMPA/kainate receptors in cultured
murine hippocampal neurones by protein kinase C", J. Physiol.,
1994, vol. 475.3, pp. 431-437. cited by applicant .
Yakel, et al., "Identification of Ca2+/calmodulin-dependent protein
kinase II regulatory phosphorylation site in
non-N-methyi-D-aspartate glutamate receptors", Proc. Nat. Acad.
Sci., Feb. 1995, vol. 92, pp. 1376-1380. cited by applicant .
Soderling, et al., "Structure and regulation of
calcium/calmodulin-dependent protein kinases II and IV", Biochimica
et Biophysica Acta, 1996, vol. 1297, pp. 131-138. cited by
applicant .
Barria, et al., "Identification of the Ca2+/Calmodulin-dependent
Protein Kinase II Regulatory Phosphorylation Site in the
alpha-Amino-3-hydroxyl-5-methyl-4-isoxazole-propionate-type
Glutamate Receptor", J. Bioi. Chern. Dec. 26, 1997, vol. 272, No.
52, pp. 32727-32730. cited by applicant .
Xia, et al., "Clustering of AMPA Receptors by the Synaptic PDZ
Domain-Containing Protein PICK1 ", Neuron, Jan. 1999, vol. 22, pp.
179-187. cited by applicant .
Dong, et al., "GRIP: a synaptic PDZ domain-containing protein that
interacts with AMPA receptors", Nature, Mar. 20, 1997, vol. 386,
pp. 279-284. cited by applicant .
Osten, et al., "The AMPA Receptor GluR2 C Terminus Can Mediate a
Reversible, AIP-Dependent Interaction with NSF and alpha- and
beta-SNAPs", Neuron, Jul. 1998, vol. 21, pp. 99-110. cited by
applicant .
Daw, et al., "PDZ Proteins Interacting with C-Terminal GluR2/3 Are
Involved in a PKC-Dependent Regulation of AMPA Receptors at
Hippocampal Synapses", Neuron, Dec. 2000, vol. 28, pp. 873-886.
cited by applicant .
Allison, et al., "Role of Actin in Anchoring Postsynaptic Receptors
in Cultured Hippocampal Neurons: Differential Attachment of NMDA
versus AMPA Receptors", J. Neurosci., Apr. 1, 1998, vol. 18(7), pp.
2423-2436. cited by applicant .
O'Brien, et al., "Synaptic Clustering of AMPA Receptors by the
Extracellular Immediate-Early Gene Product Narp", Neuron, Jun.
1999, vol. 23, pp. 309-323. cited by applicant .
Chuang, et al., "Glyceraldehyde-3-Phosphate Dehydrogenase,
Apoptosis, and Neurodegenerative Diseases", Annu. Rev. Pharmacal.
Toxicol., 2005, vol. 45, pp. 269-290. cited by applicant .
Sirover, "New Nuclear Functions of the Glycolytic Protein,
Glyceraldehyde-3-Phosphate Dehydrogenase, in Mammalian Cells",
Journal of Cellular Biochemistry, 2005, vol. 95, pp. 45-52. cited
by applicant .
Sawa, et al., "Giyceraldehyde-3-Phosphate dehydrogenase: Nuclear
translocation participates in neuronal and nonneuronal cell death",
Proc. Natl. Acad. Sci., Oct. 14, 1997, vol. 94, No. 21, pp.
11669-11674. cited by applicant .
Ishitani, et al., "Nuclear Localization of Overexpressed
Glyceraldehyde-3-Phosphate Dehydrogenase in Cultured Cerebellar
Neurons Undergoing Apoptosis", Mol. Pharmacal., 1998, vol. 53, pp.
701-707. cited by applicant .
Ishitani, et al., "Giyceraldehyde-3-phosphate dehydrogenase
antisense oligodeoxynucleotides protect against cytosine
arabinonucleoside-induced apoptosis in cultured cerebellar
neurons", Proc. Natl. Acad. Sci., Sep. 3, 1996, vol. 93, No. 18,
pp. 9937-9941. cited by applicant .
Hara, et al., "S-nitrosylated GAPDH initiates apoptotic cell death
by nuclear translocation following Siah1 binding", Nature Cell
Biology, Jul. 2005, vol. 7, No. 7, pp. 665-674. cited by applicant
.
Tsai, et al., "Studies on a Mammalian Cell Protein (P8) with
Affinity for DNA in vitro", J. Mol. Bioi., 1973, vol. 73, pp.
307-316. cited by applicant .
Singh, et al., "Sequence-Specific Binding of Transfer RNA by
Glyceraldehyde-3-Phosphate Dehydrogenase", Science, Jan. 15, 1993,
vol. 259, No. 5093, pp. 365-368. cited by applicant .
Baxi, et al., "Uracil DNA-Giycosylase/Giyceraldehyde-3-Phosphate
Dehydrogenase is an Ap4A Binding Protein", Biochemistry, 1995, vol.
34, No. 30, pp. 9700-9707. cited by applicant .
Nagy, et al., Giyceraldehyde-3-phosphate Dehydrogenase Selectively
Binds AU.cndot.rich RNA in the NAD+-binding Region (Rossmann Fold),
J. Bioi. Chern., Feb. 10, 1995, vol. 270, No. 6, pp. 2755-2763.
cited by applicant .
Schultz, et al., "Specific Interaction of Glyceraldehyde
3-Phosphate Dehydrogenase with the 5' -Nontranslated RNA of
Hepatitis A Virus", J. Bioi. Chern., Jun. 14, 1996, vol. 271, No.
24, pp. 14134w14142. cited by applicant .
Tisdale, "Giyceraldehyde-3-phosphate Dehydrogenase Is Required for
Vesicular Transport in the Ear1y Secretory Pathway", J. Bioi.
Chern., Jan. 26, 2001, vol. 276, No. 4, pp. 2480.cndot.2486. cited
by applicant .
Tisdale, "Giyceraldehyde-3-phosphate Dehydrogenase Is
Phosphorylated by Protein Kinase Ciota Ilamda and Plays a Role in
Microtubule Dynamics in the Ear1y Secretory Pathway", J. Bioi.,
Chern., Feb. 1, 2002, vol. 277, No. 5, pp. 3334w3341. cited by
applicant .
Tisdale, "Giyceraldehyde-3-phosphate Dehydrogenase Interacts with
Rab2 and Plays an Essential Role in Endoplasmic Reticulum to Golgi
Transport Exclusive of Its Glycolytic Activity", J. Bioi. Chern.,
Dec. 24, 2004, vol. 279, No. 52, pp. 54046-54052. cited by
applicant .
Kumagi, et al., "A Porcine Brain Protein (35 K Protein) which
bundles Microtubules and Its Identification as Glyceraldehyde
3-Phosphate Dehydrogenase", J. Biochem., 1983, vol. 93, No. 5, pp.
1259-1269. cited by applicant .
Glaser, et al., "Tubulin is the endogenous inhibitor of the
glyceraldehyde 3-phosphate dehydrogenase isoform that catalyzes
membrane fusion: Implications for the coordinated regulation of
glycolysis and membrane fusion", Oct. 29, 2002, vol. 99, No. 22,
pp. 14104-14109. cited by applicant .
Chen, et al., "Involvement of Glyceraldehyde-3.cndot.Phosphate
Dehydrogenase (GAPDH) and p53 in Neuronal Apoptosis: Evidence that
GAPDH is Upregulated by p53", J. Neurosci., Nov. 1, 1999, vol.
19(21), pp. 9654-9662. cited by applicant .
Dastoor, et al, "Potential role of nuclear translocation of
glyderaldehyde-3-phosphate dehydrogenase in apoptosis and oxidative
stress", Journal of Cell Science, 2001, vol. 114 (9), pp.
1643-1653. cited by applicant .
Barbosa, et al., Glyceraldehyde-3-Phosphate Dehydrogenase of
Paracoccidioides brasiliensis Is a Cell Surface Protein Involved in
Fungal Adhesion to Extracellular Matrix Proteins and Interaction
with Cells, Infection and Immunity, Jan. 2006, vol. 74, No. 1, pp.
382-389. cited by applicant .
Bhatiacharya, et al, "Localization of Functional Prostaglandin E2
Receptors EP3 and EP4 in the Nuclear Envelope", The Journal of
Biological Chemistry, May 28, 1999, vol. 274, No. 22, pp.
15719-15724. cited by applicant .
Bkaily, et al., "Presence of Functional Endothelin-1 Receptors in
Nuclear Membranes of Human Aortic Vascular Smooth Muscle Cells",
Journal of Cardiovascular Pharmacology, 2000, vol. 36 (Suppl. 1),
pp. S414-S417. cited by applicant .
Brooks, et al., "Ubiquitination, phosphorylation and acetylation:
the molecular basis for p53 regulation". Current Opinion in Cell
Biology, 2003, vol. 15, pp. 164-171. cited by applicant .
Carriedo, et al., "Rapid Ca2+ Entry through Ca2+ .cndot.Permeable
AMPA/Kainate Channels Triggers Marked Intracellular Ca2+ Rises and
Consequent Oxygen Radical Production", J. Neurosci., Oct. 1, 1998,
vol. 18(19), pp. 7727 .cndot. 7738. cited by applicant .
Carroll, et al, "Dynamin-<lependent endocytosis of ionotropic
glutamate receptors", Proc. Natl. Acad. Scie., Nov. 23, 1999, vol.
96, No. 24, pp. 14112-14117. cited by applicant.
|
Primary Examiner: Ulm; John
Claims
What is claimed is:
1. An excitotoxicity-inhibiting polypeptide of between 30 and 200
amino acids comprising a first amino acid sequence defined by SEQ
ID NO:2, and a second amino acid sequence comprising a binding
domain wherein the second amino acid sequence is heterologous to
said first amino acid sequence.
2. The polypeptide of claim 1, wherein said binding domain
comprises a protein transduction domain which is heterologous to
said first amino acid sequence.
3. The polypeptide of claim 1, wherein said polypeptide is attached
covalently to a non-protein substrate.
4. The polypeptide of claim 3, wherein the polypeptide or
non-protein substrate is labeled.
5. A kit comprising the polypeptide defined by claim 1, one or more
diluents, delivery vehicles, or pharmaceutically acceptable
excipients and one or more devices for delivering polypeptides to a
solution, cell, cell culture, tissue, organ or subject.
6. A composition comprising the excitotoxicity-inhibiting
polypeptide of claim 1 and one or more diluents, delivery vehicles,
or pharmaceutically acceptable excipients.
7. A composition comprising the excitotoxicity-inhibiting
polypeptide of claim 1.
8. A method of inhibiting AMPA receptor-mediated excitotoxicity
comprising, administering, a polypeptide comprising to a cell,
tissue or subject comprising cells exhibiting GAPDH-P53 interaction
and in need thereof.
9. The method of claim 8, wherein said method is practiced in a
subject in vivo.
10. The method of claim 9, wherein the subject is a human
subject.
11. The method of claim 10, wherein the human subject has or is at
risk of an ischemic event or a condition characterized by glutamate
accumulation.
12. A method of inhibiting GAPDH association with p53 comprising
administering a polypeptide comprising 30-200 amino acids and
comprising SEQ ID NO:2, to a solution, cell, cell culture, tissue
or subject comprising GAPDH and p53.
13. A method of treating or preventing brain injury associated with
an ischemic event or a condition characterized by glutamate
accumulation comprising, administering, a polypeptide comprising
SEQ ID NO:2 to a subject in need thereof.
Description
FIELD OF INVENTION
The present invention relates to compositions and methods for
modulating AMPA receptor-mediated excitotoxicity.
BACKGROUND OF THE INVENTION
Ischemic stroke is a worldwide public health problem and one of the
leading causes of death in humans. A role for
excitotoxicity-mediated by glutamate receptors has stimulated
intensive research for decades. This has led to the hope that
compounds antagonizing the glutamate receptor function may be of
clinical benefit in treating stroke. However, the antagonist
therapy failed in stroke trials, in most cases because of a limited
therapeutic window and severe side effects, caused by the essential
requirement of glutamate receptor-mediated excitatory
neurotransmission in maintaining normal brain function.
Glutamate is the principal excitatory neurotransmitter in the brain
and is involved in numerous physiological functions and processes
including neuronal circuit development, learning and memory, as
well as with many neuropathological disorders, such as the
neurotoxicity associated with stroke. Glutamate activates two major
subfamilies of ligand-gated postsynaptic receptors: AMPA
(.alpha.-amino-3-hydroxyl-5-methyl-4-isoxazolepropionic acid)
receptor and NMDA (N-methyl-D-aspartate) receptor (1). AMPA
receptors mediate most of the excitatory postsynaptic current at
resting membrane potentials while NMDA receptors are critically
important in producing a number of different forms of synaptic
plasticity in AMPA receptor-mediated synaptic transmission (2).
Glutamate accumulation, in pathological condition such as
immediately after ischemia, results in extensive stimulation of its
receptors which can be highly neurotoxic (3,4). NMDA
receptor-mediated neurotoxicity is dependent on extracellular Ca2+
and thus may reflect a large amount of Ca2+ influx directly through
the receptor-gated ion channels (3,4). Most models of ischemic
neurodegeneration have focused on the putative role of NMDA
receptor activation. However, use of NMDA antagonists in animal
models of ischemia as well as in human clinical trials has not
generally shown the anticipated robust efficacy (5), suggesting
NMDA receptor over activation may not be the sole player in the
glutamate receptor-mediated neurotoxicity. AMPA receptors has been
tightly associated with the selective pattern of neuronal loss in
certain identifiable subsets of neurons observed in transient
forebrain ischemia (6-13). However, as most AMPA receptor channels
are much less Ca2+ permeable, the mechanism linking AMPA receptor
activation to neuronal cell death remains largely unknown.
Functional changes in AMPA receptors are most often attributed to
phosphorylation and de-phosphorylation by PKA (cyclic AMP-dependent
protein kinase), protein kinase C(PKC) and CaM kinase II
(calcium-calmodulin kinase II) (14-18). Recently, a variety of
intracellular proteins have been reported to bind directly to AMPA
receptors (19-23). These proteins play important roles not only in
receptor targeting or clustering, but also in the modulation of
receptor activity and activation of signaling pathways. One recent
study reports that an extracellular secreted protein NARP binds to
the extracellular N-terminus (NT) of AMPA receptors and plays a
role in the induction of AMPA receptor clustering (24). This
contrasts with all other identified AMPA interacting proteins that
bind to the intracellular carboxyl tail (CT) of the AMPA receptor
subunits.
Molecular Biology and Functions of GAPDH:
GAPDH is a tetrameric protein (144 kDa) composed of four identical
subunits (37 kDa). The monomer is about 333-335 amino acids long,
and each monomer has binding sites for the substrate
(glyceraldehyde-3-phosphate, G-3-P) and co-factor nicotinamide
adenine dinucleotide (NAD+) (25-26). Residues 0-149 from N-termini
comprise the NAD+ binding domain; and, side chains involved in
catalysis are contained in residues from 149-333 or 149-335. The
co-factor binds reversibly to the enzyme prior to the substrate
binding.
Traditionally, GAPDH has been considered the key enzyme in
glycolysis, with a critical role in energy production. It is
considered to be the product of a housekeeping gene whose
transcript level remains constant under most of experimental
conditions. However, recent evidence supports the notion that GAPDH
plays a critical role in apoptosis during which its expression and
subcellular localization is altered (27-30). The cellular
localization of GAPDH is not only restricted to the cytosol but it
is also found in the nucleus and plasma membrane.
In the nucleus, GAPDH has been shown to act as a DNA binding
protein and t-RNA transport protein which plays a specific role in
the transportation and maintenance of nucleic acid. GAPDH binds to
and transports t-RNA from the nucleus to the cytosol, and the
interaction of GAPDH with t-RNA is displaced by the co-factor,
NAD+(31-32). In addition, the uracil DNA glycosylase activity of
GAPDH, together with its binding to diadenosine tetraphosphate
(Ap4A), imply that nuclear GAPDH is involved in DNA replication and
repair (33).
In the cytosol, RNA/GAPDH interactions enable GAPDH to play an
important role in translational regulation of gene expression by
controlling rate of protein synthesis and/or by altering the
stability of mRNA (34-35). Furthermore, GAPDH is essential for ER
to Golgi transport through its interaction with Rab2 GTPase and
atypical protein kinase C/(aPKC/), two important proteins involved
in the early secretory pathway and vesicle formation (36-38).
The function of membrane-associated GAPDH is to bind to tubulin
thereby regulating polymerization and bundling of microtubules near
the cell membrane, suggesting that GAPDH is involved in the
re-organization of sub-cellular organelles (39). Furthermore,
release of tubulin from membrane-associated GAPDH facilitates the
fusion of vesicles to the plasma membrane (40). Thus, GAPDH is
involved in both maintenance of membrane trafficking and the
promotion of vesicle fusion through modulation of cytoskeleton
functions.
GAPDH and Apoptosis:
GAPDH is overexpressed and accumulated in the nucleus during
apoptosis induced by a variety of insults. Evidence shows that the
GAPDH nuclear translocation is essential for the apoptotic cascade
(41-42). Western blot analysis and confocal immunocytochemistry
results indicate a significant increase of GAPDH expression in the
nuclear fraction subjected to various stresses. Antisense
oligonucleotides that deplete GAPDH prevent this nuclear
translocation and reduce apoptosis (41, 43-44).
The mechanism underlying GAPDH nuclear translocation and subsequent
cell death remains largely unknown, however, recent studies have
suggested several potential factors/pathways that maybe involved in
the process: the expression of GAPDH is regulated by p53, the tumor
suppressor protein and by proapoptotic transcription factor. Thus,
GAPDH could be one of the downstream apoptotic mediators (45); over
expression of bcl-2 blocks the apoptotic insults triggered by GAPDH
over expression, nuclear translocation and subsequent apoptosis,
suggesting that Bcl-2 may participate in the regulation of GAPDH
nuclear translocation. This effect may be part of the mechanism of
Bcl-2-induced protection against apoptosis (46) and GAPDH binds to
a nuclear localization signal containing protein, Siah which
initiates its translocation to the nucleus. The association with
GAPDH stabilizes Siah and thereby enhances Siah-mediated
proteolytic cleavage of its nuclear substrates, such as N-CoR and
triggers apoptosis (44, 47-49).
Molecular Biology of AMPA Receptors:
AMPA receptors are intrinsic ion channels comprised of different
subunits, which are encoded by four gene products, termed GluR1, 2,
3 and 4 (50-54). AMPA receptors are believed to exist as
heteromeric assemblies of these subunits. Each subunit posses an
extracellular NT domain, four putative transmembrane (TM) domains
of which the second is believed to be a reentrant loop, as well as
an intracellular CT domain (55-56). It is thought that the M2 loop
participates in the formation of the ion channel pore. Two 150
amino-acid sequences (termed as S1 and S2) which are separated by
the M1-M3 membrane domains appear to represent the agonist
recognition sites (57). The molecular determinant of the calcium
permeability is localized to the single amino acid in TM 2 region.
A positively charged arginine (R) residue is found in position 586
for GluR2 whereas a neutral glutamine (Q) is found in the same
position of GluR1, GluR3 and GluR4 subunits. Recombinant AMPA
receptors lacking GluR2 show high calcium permeability and
current-voltage relationships that doubly rectify (58). All four
AMPA receptor subunits occur in two alternatively spliced versions,
flip and flop. Flip differs flop version in the profile of
desensitization and these variants show differing regional
distributions which vary during development (59-60). The exact
subunit composition of native AMPA receptors is not clear, but
immunoprecipitation strategies have shown two major complexes
composed of GluR2 together with either GluR1 or GluR3 in rat
hippocampus (61). The presence of GluR2 subunit greatly reduces
Ca++ and Zn++ permeability (58, 62-65) as well as the single
channel conductance (66) of these receptors. Hence, most of AMPA
receptors at the hippocampal synapses are Ca++ and Zn++-impermeable
(62, 67-68).
AMPA receptor interacting proteins and their function: Using yeast
a two-hybrid system with the CT domain of GluR2 subunit as bait,
GRIP (Glutamate Receptor Interacting Protein, also known as AMPA
receptor-binding protein, ABP) was the first protein identified as
an AMPA receptor interacting protein (20). This finding was
followed by extensive efforts to identify other AMPA receptor
interacting proteins. Ban 4.1 and PKCy interact with both GluR1 and
GluR4 subunits (69-70); SAP97 (synapse-associated protein-97)
couples only with GluR171; GRIP1, 2 and PICK1 (protein interacting
with C kinase) bind to GluR3 and GluR4c (19,77). Also, three
additional proteins, Stargazin, NARP (neuronal activity-regulated
pentraxin), and AP2 (adaptor protein-2) bind to all of the AMPA
receptor subunits (24, 72-73).
Interactions with the GluR2 subunit of AMPA receptors are of
considerable interest due to the key biophysical properties
conferred by the presence of this subunit. GRIP1, 2, PICK1, and NSF
(N-ethylmaleimide-sensitive factor) are identified as GluR2
interacting proteins (20-21, 74-77). Two distinct interaction
domains have been identified for the GluR2 C-terminus. NSF protein
binds to a more proximal site (74,76), while the proteins GRIP1,
ABP, and PICK1 associate with the PDZ-binding motif at the very
distal end of the C-terminus (19-20, 76).
AMPA receptor interacting proteins may regulate these receptors in
a variety of ways, such as altering AMPA receptor localization,
clustering and/or trafficking. The binding of GluR2/3/4 to PICK1 is
involved in the clustering of AMPA receptors (19,77), while the
binding of GluR2/4 with NSF likely regulates rapid turnover of
synaptic receptors (21, 74-75). Disruption of GluR2/3-GRIP
interactions causes an increase in synaptic currents and prevents
the generation of LTD22 and interaction with F-- actin also plays a
role in location of AMPA receptor clusters (78).
GluR2 subunit trafficking: Understanding the mechanism controlling
surface expression of AMPA receptors in insult-vulnerable neurons
is important because 98% of these receptors are localized at the
synapse (hippocampus) (79-80) and the modulation of membrane
receptor expression is an efficient mechanism for regulating the
efficacy of synaptic transmission (80-98). AMPA receptors are
trafficked between the plasma membrane and the intracellular
compartments via delivery (insertion) and internalization
(endocytosis) pathways. Native AMPA receptors undergo
clathrin-dependent constitutive and regulated internalization
involving adaptor protein-2 (AP2) and dynamin (99-100).
Constitutive internalization counteracts constitutive receptor
insertion, ensuring a constant number of cell surface AMPA
receptors. Both receptor phosphorylation and GluR2 interacting
proteins play an important role in trafficking of these receptors.
Furthermore, NMDA receptor activity can regulated both AMPA
receptor membrane insertion and internalization and this is
important in certain forms of synaptic plasticity (100) as well as
in NMDA-mediated neuronal apoptosis (101).
Glutamate mediated neurotoxicity is thought to contribute to
neurodegeneration following a wide range of neurological insults
including ischemia, trauma, hypoglycemia and epileptic seizure
(3,4). It is believed that elevation of the extracellular glutamate
after cerebral ischemia plays a critical role in the
patho-physiological processes leading to death of ischemic brain
tissue (102-103). Excessive glutamate, through an action on mainly
on NMDA and AMPA glutamate receptors, facilitates Ca2+ influx,
which under pathological conditions can result in excitotoxicity.
The "calcium overload" hypothesis is the prominent theory
explaining excitotoxicity (4). The molecular mechanisms underlying
NMDA-mediated excitotoxicity involve many Ca2+-regulated processes
in the cell including activation of proteases (104), endonucleases
(105), nitric oxide synthase (106), the production of free radicals
(107) and mitochondrial membrane permeability (108). The "calcium
theory" can also apply to the Ca2+ permeable AMPA receptor-induced
toxicity, however, there must be another explanation for the
Ca2+-inpermeable AMPA receptor induced toxicity. One possibility
for Ca2+-impermeable AMPA receptor induced toxicity is to induce
membrane depolarization via Na+ influx. The AMPA-mediated
depolarization, in turn, opened both VSCCs and removed the Mg2+
block from NMDA receptors, thus allowing Ca2+ influx through these
pathways (109-110). Another possibility is that AMPA
receptor-mediated ion fluxes could be coupled to downstream
neurotoxic second messengers via interactions with submembrane
proteins. For example, the interaction of GRIP1 with GRASP-1 may
couple AMPA receptors to Ras signaling (111) and GRASP-1 has been
shown to be a neuronal substrate for caspase-3 (111) which is
cleaved in apoptotic neurons in a time-dependent manner during
development and ischemia (112). Furthermore, the potential role of
GluR2-interacting proteins in excitotoxicity may be that the
presence of GluR2 is required to maintain synaptic structure and
organization. Accordingly, the toxicity observed in GluR2-deficient
neurons may result from the effects on synaptic organization and
function rather than due to AMPA receptor Ca2+ permeability. An
interesting candidate protein is the NSF, as it has been shown both
to interact with GluR2 and to mediate membrane-fusion events
(113-115). Interestingly, NSF expression increases following an
ischemic insult (116). It is not yet clear whether an increase in
NSF leads to an increase of surface expression of existing
GluR2-containing AMPA receptors following ischemia. If so, one may
speculate that increased GluR2 surface expression may decrease Ca2+
permeability through AMPA receptors, and restore synaptic
organization. Taken together, these activities indicate AMPA
receptor interacting protein may play an important role in AMPA
receptor-medited neurotoxicity.
The "GluR2 hypothesis" in AMPA receptor-mediated neurotoxicity
(117-121) predicts that a relative reduction in the expression of
GluR2 results in enhanced Ca2+-influx through newly synthesized
AMPA receptors, thereby increasing neurotoxicity; and enhancing
GluR2 membrane expression may provide protective effect based on
the evidence showing that: (1) in ischemic CA1 neurons AMPA
receptor-mediated EPSCs show an increased sensitivity to
N-(4-hydroxyphenylpropanoyl)-spermine (NHPP-spermine) (122-123), a
selective blocker for GluR2-lacking AMPA receptors (124-125).
Indicative of a reduction in the number of GluR2 containing
receptors; ischemic insults promote internalization of
GluR2-containing AMPA receptors from synaptic sites and facilitate
delivery of GluR2-lacking AMPA receptor (126); GluR2 expression is
down regulated in vulnerable neurons in animal models of transient
forebrain ischemia and epilepsy (127) and vulnerable CA1 pyramidal
neurons can be rescued from forebrain ischemic injury by enhancing
the expression of GluR2 containing receptors (127-128).
This evidence indicates the role of GluR2 membrane expression in
the AMPA receptor-mediated neurotoxicity, which raise the
possibility for proteins that regulate GluR2 subunit trafficking
through protein-protein interaction with GluR2 to be involved in
the AMPA receptor mediated apoptosis.
There is a need in the art for compositions and methods for
modulating AMPA receptor-mediated excitotoxicity. There is also a
need in the art for compositions and methods for modulating GAPDH
association with GluR2 subunit or p53.
SUMMARY OF THE INVENTION
The present invention relates to compositions and methods for
modulating AMPA receptor-mediated excitotoxicity.
According to the present invention there is provided an
excitotoxicity-inhibiting polypeptide comprising an amino acid
sequence that modulates Glu-R2-containing AMPA receptor signal
transduction, wherein said polypeptide does not encompass a
naturally occurring GluR2 subunit or GAPDH polypeptide.
Also provided by the present invention is an
excitoxicity-inhibiting polypeptide as defined above, comprising an
amino acid sequence selected from the group consisting of:
a) GluR2 NT1-3-2 (Y142-K172) amino acid sequence (SEQ ID NO:1), or
a sequence which is at least 80% identical to SEQ ID NO:1 that
binds to GAPDH and wherein said polypeptide does not encompass a
naturally occurring full length GluR2 subunit polypeptide, and;
b) GAPDH(2-2-1-1) (I221-E250) amino acid sequence (SEQ ID NO:2), or
a sequence which is at least 80% identical to SEQ ID NO:2 that
binds to p53 and wherein said polypeptide does not encompass a
naturally occurring full length GAPDH polypeptide.
Also provided by the present invention is an
excitoxicity-inhibiting polypeptide as defined above, comprising
the GluR2 NT1-3-2 (Y142-K172) amino acid sequence (SEQ ID NO:
1).
Also provided by the present invention is an
excitoxicity-inhibiting polypeptide as defined above, comprising
the GAPDH(2-2-1-1) (I221-E250) amino acid sequence (SEQ ID
NO:2).
Also provided by the present invention is an
excitoxicity-inhibiting polypeptide as defined above, wherein the
polypeptide is a fusion protein.
Also provided by the present invention is an
excitoxicity-inhibiting polypeptide as defined above, wherein the
fusion protein comprises a protein transduction domain.
Also provided by the present invention is an
excitoxicity-inhibiting polypeptide as defined above, the
polypeptide attached covalently or non-covalently to a non-protein
substrate, non-protein molecule, non-protein macromolecule, a
support, or any combination thereof. Further, the polypeptide,
non-protein substrate, non-protein molecule, non-protein
macromolecule, support or any combination thereof may be
labeled.
The present invention also provides a nucleic acid encoding the
excitotoxicity-inhibiting polypeptide as defined above.
The present invention also provides a method of inhibiting AMPA
receptor-mediated excitotoxicity comprising, administering, a
polypeptide comprising the GluR2 NT1-3-2 (Y142-K172) amino acid
sequence (SEQ ID NO:1) or GAPDH(2-2-1-1) (I221-E250) amino acid
sequence (SEQ ID NO:2) or a nucleic acid capable of expressing a
polypeptide comprising the GluR2 NT1-3-2 (Y142-K172) amino acid
sequence (SEQ ID NO:1) or GAPDH(2-2-1-1) (I221-E250) amino acid
sequence (SEQ ID NO:2), to a cell, tissue or subject in need
thereof.
Also according to the present invention is a method as defined
above wherein the wherein the method is practiced in a subject in
vivo.
Also according to the present invention is a method as defined
above, wherein the subject is a human subject. Further, the human
subject may have or be at risk of stroke, epilepsy, traumatic brain
injury, brain damage resulting from cardiac bypass surgery or a
combination thereof.
Also provided by the present invention is a method of inhibiting
GAPDH association with either the GluR2 subunit or p53 comprising
administering a polypeptide comprising the GluR2 NT1-3-2
(Y142-K172) amino acid sequence (SEQ ID NO:1) or GAPDH(2-2-1-1)
(I221-E250) amino acid sequence (SEQ ID NO:2), to a solution, cell,
cell culture, tissue or subject comprising GAPDH and either GluR2
subunit or p53.
Also provided by the present invention is a method of treating or
preventing brain injury associated with stroke, epilepsy, trauma,
cardiac bypass surgery or a combination thereof comprising,
administering, GluR2 NT1-3-2 (Y142-K172) amino acid sequence (SEQ
ID NO:1) or GAPDH(2-2-1-1) (I221-E250) amino acid sequence (SEQ ID
NO:2) or a nucleic acid capable of expressing a polypeptide
comprising the GluR2 NT1-3-2 (Y142-K172) amino acid sequence (SEQ
ID NO:1) or GAPDH(2-2-1-1) (I221-E250) amino acid sequence (SEQ ID
NO:2), to a subject in need thereof.
Also provided by the present invention is a kit comprising, a) a
polypeptide comprising the GluR2 NT1-3-2 (Y142-K172) amino acid
sequence (SEQ ID NO:1), b) a nucleic acid capable of expressing a
polypeptide comprising the GluR2 NT1-3-2 (Y142-K172) amino acid
sequence (SEQ ID NO:1), c) a polypeptide that comprises GAPDH
(2-2-1-1) amino acid sequence (SEQ ID NO:2), d) a nucleic acid
capable of expressing a polypeptide comprising the GAPDH (2-2-1-1)
amino acid sequence (SEQ ID NO:2), e) one or more diluents,
delivery vehicles, pharmaceutically acceptable excipients, or a
combination thereof, f) one or more devices for delivering
polypeptides or nucleic acids to a solution, cell, cell culture,
tissue, organ or subject, g) instructions for using any component
in the kit or practicing any method as described herein, or any
combination or sub-combination thereof.
The present invention also provides a composition comprising the
excitotoxicity-inhibiting polypeptide as defined above and one or
more diluents, delivery vehicles, pharmaceutically acceptable
excipients, or a combination thereof. Further, the composition may
comprise polypeptides independently comprising SEQ ID NO:1 and SEQ
ID NO:2. Also contemplated are compositions comprising one or more
diluents, delivery vehicles, pharmaceutically acceptable
excipients, or a combination thereof.
This summary of the invention does not necessarily describe all
features of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
These and other features of the invention will become more apparent
from the following description in which reference is made to the
appended drawings wherein:
FIG. 1 shows nucleotide and amino acid sequences of polypeptides
and nucleic as described herein. (A) shows the GluR2NT1-3-2
(Y142-K172) amino acid sequence (SEQ ID NO:1). (B) shows the
GAPDH(2-2-1-1) (I221-E250) amino acid sequence (SEQ ID NO:2). (C)
shows a representative nucleotide sequence encoding a polypeptide
that comprises the GluR2 NT1-3-2 (Y142-K172) amino acid sequence
(SEQ ID NO:3). The shaded and underlined regions show a nucleotide
sequence encoding residues Y142 to K172. (D) shows a polypeptide
sequence of GluR2 comprising V22 to E545 (SEQ ID NO:4). The GluR2
NT1-3-2 (Y142-K172) amino acid sequence is underlined. (E) shows
the amino acid sequence of GAPDH (SEQ ID NO:5) from Homo sapiens.
The GAPDH(2-2-1-1) (I221-E250) sequence is underlined.
FIG. 2 shows Coomassie blue stained SDS-PAGE gel of the protein
selectively pulled down by GST-GluR2NT. Positions of molecular size
are shown. Protein of interest: .about.37 kDa.
FIG. 3 shows biochemical association of the GluR2 subunit with
GAPDH. (A) Communoprecipitation of GAPDH from solubilized rat
hippocampal lysates by GluR2 subunit (antibody). (B) Detergent
extracts of rat hippocampus were incubated with GST-fusion proteins
of GluR2CT or GluR2NT coupled to Glutathione-Sepharose beads for
affinity purification. The eluted bound proteins were loaded on 10%
SDS-PAGE gel and immunoblotted with primary antibody anti-GAPDH.
IP, Immunoprecipitation.
FIG. 4 shows identification of the GluR2 subunit region involved in
the GAPDH and GluR2 NT interaction. (A) Schematic representation of
the generated GluR2 NTa; GluR2 NTb; GluR2 NTc mini-genes. (B)
Western blotting of GAPDH from solubilized rat hippocampal extracts
showed the presence of GAPDH after affinity precipitation by
GST-GluR2 NTa, but not by GST-GluR2 rub, GST-GluR2 NTc or GST
alone. (C) The [35S]-GAPDH probe bound with GST-GluR2 NTa, but not
with GluR2 NTb, GluR2 NTc or GST alone in vitro binding assay.
FIG. 5 shows identification of the GluR2 subunit region involved in
the GAPDH and GluR2 NT interaction. (A) Schematic representation of
the generated GluR2 NTa1; GluR2 NTa2; GluR2 NTa3; GluR2 NTa4 and
GluR2 NTa5 mini-genes. (B) Western blotting of GAPDH from
solubilized rat hippocampal extracts showed the presence of GAPDH
after affinity precipitation by GST-GluR2 NTa3, but not by others
or GST alone. (C) [35S]-GAPDH probe bound with GST-GluR2 NTa3, but
not with others or GST alone In vitro binding assay.
FIGS. 6 (A-C) show identification of the GluR2 subunit region
involved in the GAPDH and GluR2NT interaction. (A) Schematic
representation of the generated GluR2 NTa3-1 and GluR2 NT1-3-2
mini-genes. (B) Western blotting of GAPDH from solubilized rat
hippocampal extracts showed the presence of GAPDH after affinity
precipitation by GST-GluR2 NT1-3-2, but not by GST-GluR2 NTa3-1 or
GST alone. (C) [35S]-GAPDH probe bound with GST-GluR2 NT1-3-2, but
not with GST-GluR2 NTa3-1 or GST alone in vitro binding assay.
FIGS. 6(D-L) show identification and validation of the GluR2 region
involved in the GAPDH-GluR2 interaction. (D), Coomassie blue
stained SDS-PAGE gel of the protein selectively pulled down by
GST-GluR2.sub.NT, but not GluR1.sub.NT or GST alone from
solubilized rat hippocampal lysate (20 .mu.g GST peptide, 100 .mu.g
of hippocampal tissue). Positions of molecular size are shown.
Protein of interest: .about.37 kDa. (E) GAPDH was specifically
pulled down by GST-GluR2.sub.NT (20 .mu.g) in detergent extracts of
rat hippocampus (100 .mu.g), but not GST-GluR2.sub.CT or GST alone.
(F) Schematic representation of GST-fusion proteins encoding
GluR2.sub.NT1 to GluIR2.sub.NT3, GluR2.sub.NT1-1 to
GluR2.sub.NT1-5, GluR2.sub.NT1-3-1 and GluR2 NT1-3-2. (G-I)
Affinity purification of GAPDH from solubilized rat hippocampal
tissue (100 .mu.g amount) using 20 .mu.g GST fusion peptides
encoding truncated versions of GluR2. GAPDH was specifically pulled
down by GST-GluR2.sub.NT1 (G) GST-GluR2.sub.NT1-3 (H) and GST-GluR2
NT1-3-2 (I) but not by the other GST fusion proteins or by GST
alone. (J-L) Using an in vitro binding assay, [.sup.35S]-GAPDH
probe bound with specific GST-GluR2.sub.NT1 (J).
GST-GluR2.sub.NT1-3 (K) and GST-GluR2 NT1-3-2 (L) fragments, but
not with other GST fusion proteins or GST alone.
FIG. 7 shows association of the GluR2 subunit with GAPDH in
transfected HEK 293T cells. GAPDH co-immunoprecipitated the GluR2
subunit revealing that these proteins associate without exogenous
AMPA receptor agonist stimulation. The insertion of GluR2 NT1-3-2
mini-gene interrupted the protein-protein interaction. The directly
immunoprecipitated GluR2 subunit was used as a loading control.
Rabbit IgG and rat hippocampal extracts were used as negative
control and positive control, respectively. IP,
Immunoprecipitation.
FIG. 8 shows activity-dependent association of the GluR2 subunit
with GAPDH in transfected HEK 293T cells. Application of glutamate
enhanced the protein-protein interaction, which was blocked by the
competitive AMPA receptor antagonist CNQX. The directly
immunoprecipitated GluR2 subunit was used as a loading control.
Rabbit IgG and rat hippocampal extracts were used as negative
control and positive control, respectively. IP,
Immunoprecipitation.
FIG. 9 shows association of the GluR2 subunit with GAPDH in
transfected HEK 293T cells. GAPDH and GluR2 were
co-immunoprecipitatedfrom transfected HEK 293T cells lysates in the
presence or absence of the GluR2 NT1-3-2 mini-gene, as well as with
and without glutamate treatment. The directly immunoprecipitated
GluR2 subunit was used as a loading control. Rabbit IgG and rat
hippocampal extracts were used as negative control and positive
control, respectively. RIgG, Rabbit IgG. IP,
Immunoprecipitation.
FIG. 10 shows Western blot analysis of the initial level of GluR2
subunit in transfected HEK293T cells, with and without 100 .mu.M
glutamate treatment. The total amount of proteins loaded was
indicated by a cytoskeletal protein .alpha.-tubulin. The intensity
of each protein band was quantified by densitometry (Software:
ImageJ from research Services Branch). Data were representative of
three independent experiments.
FIG. 11 shows the expression level of GAPDH in transfected HEK293T
cells, with or without 100 .mu.M glutamate treatment. The loading
amount of proteins is indicated by cytoskeleton protein
alpha-tubulin. The intensity of each protein band was quantified by
densitometry (Software: ImageJ from research Services Branch). Data
were representative of three independent experiments.
FIG. 12 shows the expression level of the GluR2 subunit and GAPDH
in different cell compartments in the presence or absence of the
GluR2 NT1-3-2 mini-gene. (A) 100 .mu.M glutamate treatment
facilitated the translocation of GAPDH, while the insertion of the
GluR2 NT1-3-2 mini-gene reversed the increase. (B) 100 .mu.M KA
treatment increased the expression of the GluR2 subunit, while the
insertion of the GluR2 NT1-3-2 mini-gene diminished this increase.
Data were representative of three independent experiments.
FIG. 13 shows interruption of the GAPDH and GluR2 interaction
resulted in an increase in the GluR2 subunit cell surface
expression following glutamate treatment in HEK-293T cells. (A) The
interruption of GAPDH and GluR2 interaction using the GluR2 NT1-3-2
mini-gene had no significant effect on the GluR2 cell surface
expression in the absence of glutamate. (B) Interruption OF the
interaction with the GluR2 NT1-3-2 mini-gene showed a significant
increase in cell surface GluR2 expression after 100 .mu.M glutamate
treatment for 30 minutes. The asterisk indicates a significant
difference from the AMPA+pcDNA3 group (p<0.05; n=9).
FIG. 14 shows results that suggest interruption of the GAPDH and
GluR2 interaction results in an increase in GluR2 subunits
localized at the cell surface after KA in hippocampal neurons. (A)
Pretreatment with 10 .mu.M TAT-GluR2 NT1-3-2 peptide to interrupt
the GAPDH and GluR2 interaction had no significant effect on the
GluR2 expression at the cell surface compared to the group
pretreated with 10 .mu.M TAT-only peptide. (B) The interruption of
the protein-protein interaction caused by pretreatment of 10 .mu.M
TAT-GluR2 NT1-3-2 peptide increased cell surface GluR2 expression
after KA treatment, compared to the TAT-only group. Data are
analyzed by Student's t test. The asterisk indicates a significant
difference from the AMPA+pcDNA3 group (p<0.05; n=9).
FIG. 15 shows results that suggest that interruption of the GAPDH
and GluR2 interaction results in an increase in GluR2 subunits
localized at the cell surface in OGD model. The interruption of the
protein-protein interaction caused by pretreatment of 10 .mu.M
TAT-GluR2 NT1-3-2 peptide increased cell surface GluR2 expression
after oxygen-glucose derivation for 2 hours when compared to the
TAT-only group. Data are analyzed by Student's t test. The double
asterisk indicates a significant difference from the OGD group
(p<0.01; n=9).
FIG. 16 shows results of regulation of the AMPA receptor-mediated
excitotoxicity in HEK293T cells expressing GluR1 and GluR2 subunits
by the insertion of GluR2 NT1-3-2 mini-gene. Quantification of AMPA
receptor-mediated excitotoxicity through quantitative measurements
of PI fluorescence after indicated treatment. After glutamate
treatment, the insertion of GluR2 NT1-3-2 mini-gene diminished cell
death when compared to the GluR2 NT1-3-2 mini-gene sham-transfected
group. Data were analyzed by student's t test. The double asterisks
indicate a significant difference from AMPAR+pcDNA group
(p<0.01; n=9)
FIG. 17 shows results that regulation of the AMPA receptor-mediated
excitotoxicity in rat hippocampal neuron culture. Quantification of
the AMPA receptor-mediated excitotoxicity through quantitative
measurements of PI fluorescence after indicated treatments is
described in the Examples. Pretreatment with 10 .mu.M TAT-GluR2
NT1-3-2 peptide reduced cell death, compared to the group
pretreated with 10 .mu.M TAT-only peptide. Data were analyzed by
student's t test. The triple asterisks indicate a significant
difference from AMPAR+pcDNA3 group (p<0.001; n=9).
FIG. 18 shows results of regulation of the AMPA receptor-mediated
excitotoxicity in the OGD model. The AMPA receptor-mediated
excitotoxicity was measured through quantitative measurements of PI
fluorescence after indicated treatments. Pretreatment with 10 .mu.M
TAT-GluR2 NT1-3-2 peptide reduced cell death when compared to the
group pretreated with 10 .mu.M TAT-only peptide. Data were analyzed
by student's t test. The asterisk indicates a significant
difference from AMPAR+pcDNA3 group (p<0.001; n=9).
FIG. 19 shows results suggesting molecules involved in the
regulation of AMPA-receptor mediated excitotoxicity. The expression
levels of PARP, P53, caspase-3, Bcl-2 and Bcl-x were tested by
immunoblotting. In transfected HEK 293T cells, glutamate treatment
(100 .mu.M) and the insertion of the GluR2 NT1-3-2 mini-gene
affected the expression level of PARP, caspase-3, Bcl-2 and
Bcl-x.
FIG. 20 shows results of biochemical association of AIF with the
GluR2 subunit and APDH. Detergent extracts of rat hippocampus were
incubated with GST-fusion proteins of GluR2CT or GluR2NT coupled to
Glutathione-Sepharose beads for affinity purification. (A) AIF was
precipitated by GST-GluR2NT, but not by GST-GluR2CT or GST alone.
(B) Western blotting of AIF from solubilized rat hippocampal
extracts showed the presence of AIF after affinity precipitation by
GST-GAPDH, but not by GST alone.
FIG. 21 shows results validating agonist regulation of the
extracellular GAPDH: AMPAR complex formation. (A)
Co-immunoprecipitation of GAPDH with the GluR2 subunit from
solubilized rat hippocampus. (B-C) Activation of AMPAR (HEK-293T:
100 .mu.M glutamate, 30 min; neurons: 100 .mu.M kainic acid [KA],
30 min), enhanced the association of GAPDH and GluR2 subunit, which
was blocked by preincubation with the GluR2 NT1-3-2 peptide in both
HEK-293T cells expressing GluR1/2 subunits (B) and primary cultures
of rat hippocampal neurons (C) but did not affect directly
immunoprecipitated GluR2 levels (B, C, bottom panels). (D) Using a
rabbit anti-GAPDH antibody, GAPDH immunoprecipitated from the
conditioned medium (CM) of primary cultures of rat hippocampal
neurons but not from'fresh control medium. Rabbit IgG was used as
negative control. (E) Conditioned media of nontransfected HEK-293T
cells and HEK-293T cells transfected with GluR1/2 subunits, in the
presence or absence of glutamate (Glut), was concentrated to
examine the expression of GAPDH and .alpha.-tubulin. GAPDH was
present in conditioned media and cell lysates, while
.alpha.-tubulin was only present in cell lysates. (F) Rat
hippocampal neurons were incubated with sulfo-NHS-LC biotin to
label cell surface proteins. The amount of GAPDH that
co-immunoprecipitated with GluR2 subunit was examined in both
non-biotinylated (NB) and biotinylated (B) proteins.
FIG. 22 shows results suggesting activation of AMPAR induces GAPDH
internalization in HEK-293T cells co-expressing GluR1/GluR2
subunits. (A) Glutamate (100 .mu.M, 30 min) induced cell surface
GluR2 internalization by 26.3.+-.4.1%. t-test *Significantly
different from control group (n=9, P<0.05). (B) Agonist
stimulation induced cell surface GAPDH internalization by
20.6.+-.3.9%, while preincubation of the GluR2 NT1-3-2 peptide
abolished the agonist-induced GAPDH internalization. ANOVA,
followed by post-hoc Student-Newman-Keuls test *Significantly
different from control group; # significantly different from
glutamate group (n=9, P<0.05). (C) Glutamate failed to
internalize GAPDH in HEK-293T cells in the absence of GluR1/GluR2
subunits. (D) Glutamate failed to internalize GAPDH in HEK-293T
cells transfected with GluR1/GluR3 subunits. (E) Glutamate induced
GluR2 internalization by 18.1.+-.0.6% in HEK-293T cells transfected
with wild type dynamin, which was blocked by the co-expression of
mutant K44E dynamin. ANOVA, followed by post-hoc
Student-Newman-Keuls test *Significantly different from control
group (n=9, P<0.05). (F) The glutamate induced GAPDH
internalization by 21.4.+-.8.3% in HEK-293T cells transfected with
wild type dynamin, which was blocked by the co-expression of mutant
K44E dynamin. ANOVA, followed by post-hoc Student-Newman-Keuls test
*Significantly different from control group (n=9, P<0.05).
FIG. 23 shows results suggesting that translocation of cell surface
GAPDH and GluR2 into nucleus is dependent on the GAPDH-GluR2
interaction. (A-C) Nuclei from HEK-293T cells cotransfected with
GluR1/GluR2 were purified, solubilized and run on SDS-PAGE with
subsequent Western blot analysis. Both GAPDH and GluR2 nuclear
expression was significantly increased upon glutamate treatment
(100 .mu.M, 30 min) and the nuclear translocation could be blocked
by pretreatment with the GluR2 NT1-3-2 peptide (10 .mu.M, 1 hr).
The intensity of protein bands were measured by Image J software
and normalized to the corresponding control samples. (D-F) In
hippocampal neurons, both GAPDH and GluR2 nuclear expression was
significantly increased upon KA treatment (100 .mu.M KA, 10 .mu.M
MK-801, 2 .mu.M nimodipine, 30 min) and the nuclear translocation
could be blocked by GluR2 NT1-3-2 peptide (10 .mu.M, 1 hr). The
intensity of protein bands were measured by Image J software and
normalized to the corresponding control samples. (G) Biotinylated
cell surface GAPDH and GluR2 translocates to the nucleus. Primary
cultures of rat hippocampal neurons were labeled with biotin and
then treated with GluR2 NT1-3-2 peptides before agonist
stimulation. Nuclei were isolated and nuclear biotinylated proteins
were separated from non-biotinylated proteins. Nuclear biotinylated
proteins were then run on SDS-PAGE gels and analyzed under
subsequent Western blot analysis to examine the nuclear
localization of cell surface GAPDH and GluR2.
FIG. 24 shows results suggesting biochemical association of nuclear
GAPDH and p53. (A) p53 was specifically pulled down by GST-GAPDH
from rat hippocampal extracts, but not by GST-GluR2.sub.NT or GST
alone. (B) Co-immunoprecipitation of GAPDH with p53 from extracted
nuclear proteins of HEK-293T cells expressing AMPAR, treated with
100 .mu.M glutamate and inhibited by GluR2 NT1-3-2 peptide
pretreatment. (C) The interaction between GAPDH-GluR2 is inhibited
by the presence of recombinant p53. The ability of GST-GluR2.sub.NT
(20 .mu.g) to pull down GAPDH from nuclear extracts of HEK-293T
cells co-expressing GluR1/GluR2 subunits treated with glutamate was
examined in the presence of increasing concentration of recombinant
p53 (GST tagged). Addition of 0.5 .mu.g of GST peptide did not
affect the ability of GST-GluR2.sub.NT to pull down GAPDH. (D)
Schematic representation of GST-fusion proteins encoding truncated
GAPDH segments. (E) p53 was specifically pulled down by
GST-GAPDH.sub.2 from rat hippocampal extracts, but not by
GST-GAPDH.sub.1 or GST alone: (F) p53 was specifically pulled down
by GST-GAPDH.sub.2-2 from rat hippocampal extracts, but not by
GST-GAPDH.sub.-2-1 or GST alone. (G) p53 was specifically pulled
down by GST-GAPDH.sub.2-2-1 from rat hippocampal extracts, but not
by GST-GAPDH.sub.2-2-2 or GST alone. (H), p53 was specifically
pulled down by GST-GAPDH(2-2-1-1) from rat hippocampal extracts,
but not by other GST fusion proteins or GST alone. (I) The
expression of GAPDH(2-2-1-1) mini-gene disrupted the
co-immunoprecipitation of p53 with GAPDH in transfected HEK-293T
cells.
FIG. 25 shows results suggesting regulation of the AMPAR-mediated
cell death in HEK-293T cells. (A) Activation of AMPAR (300 .mu.M
glutamate, 25 .mu.M CTZ, 24 hr) induced significant cell death in
HEK-293T cells expressing GluR1/2. Toxicity was indexed by
measuring propidium iodide (50 .mu.g/mL) incorporation.
***Significantly different from control group (n=9, P<0.001),
t-test. (B) Depletion of calcium with 5 mM EGTA inhibited the NR
1-1a/2A NMDA receptor-mediated cell death by 38.+-.3.6%, while the
GluR1/GluR2AMPAR-mediated cell death remained intact.
*Significantly different from NR1-1a/2A without EGTA group (n=9,
P<0.05), ANOVA, followed by post-hoc Student-Newman-Keuls test.
(C) Pretreatment with GluR2 NT1-3-2 peptide (10 .mu.M, 1 hr) in
HEK-293T cells significantly attenuated AMPAR-mediated cell death
by 56.+-.1.6%. The GluR2 NT1-3-2 peptide itself showed no effect on
HEK-293T cells in the absence of glutamate treatment.
***Significantly different from glutamate group (n=9, P<0.001),
ANOVA, followed by post-hoc Student-Newman-Keuls test. (D) The
GluR2 NT1-3-2 peptide itself showed no effect on HEK-293T cells in
the absence of GluR1/GluR2 co-expression. (E) The GluR2 NT1-3-2
peptide failed to inhibit GluR1/3 AMPAR-mediated cell death. (F)
The GluR2 NT 1-3-2 peptide significantly inhibited AMPAR-mediated
cell death (100 .mu.M KA, 10 .mu.M MK-801, 2 .mu.M nimodipine, 1
hr) by 47.6.+-.3.3% in cultured rat hippocampal neurons.
***Significantly different from KA group (n=9, P<0.001), t-test.
(G) Glutamate-induced cell death was significantly inhibited by
49.8.+-.8.3% by pre-treatment of p53 antagonist cyclic (10 .mu.M, 1
hr) PFT-.alpha. in HEK-293T cells expressing GluR 1/2.
*Significantly different from 0 .mu.M group (n=9, P<0.05),
t-test. (H) Cyclic PFT-.alpha. failed to inhibit glutamate-induced
cell death in HEK-293T cells expressing GluR1/3. (I)
Glutamate-induced cell death was significantly inhibited in cells
co-expressing GluR1/2 with GAPDH(2-2-1-1) compared to cells
expressing GluR1/2. **(J) Both the expression of p53 and the
phosphorylation of p53 at serine 46 were enhanced upon agonist
stimulation in HEK-293T cells expressing GluR 1/2, but not in cells
co-expression GluR1/2 with GAPDH(2-2-1-1) mini-gene.
FIG. 26 shows results of experiments performed using mutants of
sequences as defined herein. (A) Nuclei from HEK-293T cells
cotransfected with GluR 1/GluR2 were purified, solubilized and run
on SDS-PAGE with subsequent Western blot analysis. Both GAPDH and
GluR2 nuclear expression was significantly increased upon glutamate
treatment (100 .mu.M, 30 min) and the nuclear translocation could
NOT be blocked by co-transfection of the GluR2.sub.220-238
mini-gene. GluR2.sub.220-238 is the binding site of GluR2 and
Siah1. The intensity of protein bands were measured by Image J
software and normalized to the corresponding control samples. (B)
Schematic representation of GluR2.sub.NT mutants. GluR2-M 1 94-95
KK->AA; GluR2-M2 171-172 KK->AA; GluR2-M3 187-188 KK->AA.
(C) Both GAPDH and GluR2 nuclear expression was significantly
decreased in GluR2-M2 transfected HEK293T cells upon glutamate
treatment (100 .mu.M, 30 min). (D) GluR2-M2 inhibited
glutamate-induced cell death in AMPAR transfected HEK293T cells.
**Significantly different from GluR2-WT group (n=9, P<0.01),
ANOVA, followed by post-hoc Student-Newman-Keuls test. (E) GAPDH
was immunoprecipitated by GluR2.sub.NT wild type and GluR2.sub.NT
mutants. (F) GluR2 translocated mainly on nuclear envelope, while
GAPDH translocated mainly into nucleoplasm after AMPA receptor
activation. (G-H), CO-IP of GAPDH by GluR2 subunit (upper panel)
and p53 (lower panel) in nuclear envelope and nucleoplasm of rat
hippocampal neurons.
FIG. 27 shows results confirming neuroprotective activity of
peptide GluR2 NT1-3-2 in ischemia model. Cresyl violet was used to
stain alive neurons in hippocampus region of each animal. Total
number of cresyl violet-stained nuclei in CA1 regions were
summarized. Peptide treatment after ischemia rescued 13.2% neurons
from cell death; while peptide treatment before ischemia rescued
18.2% neurons from cell death.
FIG. 28 shows results confirming the disruption of GluR2/GAPDH
formation in rat brain following an ischemic event. (A)
Coimmunoprecipitation of GAPDH by GluR2 primary antibody from rat
hippocampal extracts of sham, peptide-treated (post-ischemia) and
ischemia groups. (B) Quantification of GAPDH-GluR2
coimmunoprecipitation from sham, peptide-treated (post-ischemia)
and ischemia groups. ANOVA, followed by post-hoc SNK test.
*Significantly different from sham group (n=3 per group,
P<0.05); #significantly different from ischemia group (n=3 per
group, P<0.05). (C) Coimmunoprecipitation of GAPDH by p53 from
nuclear proteins extracted from rat hippocampus of cham,
peptide-treated (post-ischemia) and ischemia groups. (D)
Quantification of the GAPDH-p53 coimmunoprecipitation from sham,
peptide-treated (post-ischemia) and ischemia groups. *Significantly
different from sham group (n=3 per group, P<0.05). ANOVA,
followed by post-hoc SNK test. (E), Western blot analysis of GAPDH
and GluR2 nuclear expression in rat hippocampal tissues from sham,
peptide-treated (post-ischemia) and ischemia groups. Quantification
of GluR2 (F) and GAPDH (G) nuclear expression in rat hippocampal
tissues from sham, peptide-treated (post-ischemia) and ischemia
groups. **Significantly different from peptide group (n=3 per
group, P<0.01). Due to the low GluR2 levels in the control
group, data were normalized to peptide group; t-test (F). For (G),
**significantly different from sham group (n=3 per group,
P<0.01). ##significantly different from ischemia group (n=3 per
group, P<0.01); ANOVA, followed by post-hoc SNK test.
DETAILED DESCRIPTION
The present invention relates to compositions and methods for
modulating AMPA receptor-mediated excitotoxicity.
The following description is of a preferred embodiment.
Overactivation of the
.alpha.-amino-3-hydroxy-5-methylisoxazole-4-propionic acid subtype
of glutamate receptors (AMPAR) leads to excitotoxic neuronal
injuries seen in both acute brain insults including stroke and
prolonged seizure activity, yet the underlying mechanisms remain
poorly understood. Here we report that the GluR2-containing AMPAR
form a complex with extracellular glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) through a direct protein-protein interaction
between GAPDH and the amino-terminus of the GluR2 subunit. AMPAR
activation facilitates the complex formation and results in rapid
endocytosis-dependent translocation of the complex to the nucleus,
whereby GAPDH dissociates from the AMPAR and binds to nuclear p53
and activates the p53-dependent cell death pathway. Disrupting
either GAPDH-GluR2 or GAPDH-p53 interaction protects against
AMPAR-induced cell death. Thus, our results reveal a previously
unappreciated cellular signaling pathway underlying
GluR2-containing AMPAR-dependent cell death and provide novel
targets against which new therapeutics may be developed to combat
diseases involving for example, but not limited to GluR2/AMPAR
neurotoxicity.
According to the present invention, there is provided an
excitotoxicity-inhibiting polypeptide comprising an amino acid
sequence selected from the group consisting of: a) GluR2 NT1-3-2
(Y142-K172) amino acid sequence (SEQ ID NO:1), or a sequence which
is at least 80% identical to SEQ ID NO:1 that binds to GAPDH and
wherein said polypeptide does not encompass a naturally occurring
full length GluR2 subunit polypeptide, and; b) GAPDH(2-2-1-1)
(I221-E250) amino acid sequence (SEQ ID NO:2), or a sequence which
is at least 80% identical to SEQ ID NO:2 that binds to p53 and
wherein said polypeptide does not encompass a naturally occurring
full length GAPDH polypeptide.
Without wishing to be bound by theory or limiting in any manner,
the excitotoxicity-inhibiting polypeptides of the present invention
interfere with normal GluR2 subunit AMPA receptor signal
transduction activity, for example, but not limited to, by
interacting with normal physiological protein binding partners
required for normal signal transduction.
The present invention also contemplates excitotoxicity-inhibiting
polypeptides consisting of:
a) GluR2 NT 1-3-2 (Y 142-K 172) amino acid sequence (SEQ ID NO:1),
or a sequence which is at least 80% identical to SEQ ID NO:1 that
binds to GAPDH and wherein said polypeptide does not encompass a
naturally occurring full length GluR2 subunit polypeptide, and;
b) GAPDH(2-2-1-1) (I221-E250) amino acid sequence (SEQ ID NO:2), or
a sequence which is at least 80% identical to SEQ ID NO:2 that
binds to p53 and wherein said polypeptide does not encompass a
naturally occurring full length GAPDH polypeptide.
As provided above, variations of the polypeptide sequences of SEQ
ID NO:1 and SEQ ID NO:2 are contemplated herein. For example, with
respect to SEQ ID NO:1 (GluR2 NT1-3-2), but not to be considered
limiting in any manner, one or more residues at positions 3, 5, 18,
21, 22, 23, 26 or 30 of SEQ ID NO:1 may be replaced by an alternate
amino acid residue. For instance, but without wishing to be
limiting, glutamine at position 3 may be replaced by another amino
acid, for example, but not limited to lysine. Aspartic acid at
position 5 may be replaced by another amino acid, for example, but
not limited to threonine or glutamic acid. Serine at position 18
may be replaced by another amino acid, for example, but not limited
to threonine. Glutamine at position 21 may be replaced by another
amino acid, for example, but not limited to arginine. Alanine at
position 22 may be replaced by another amino acid, for example, but
not limited to valine or isoleucine. Valine at position 23 may be
replaced by another amino acid, for example, but not limited to
isoleucine. Serine at position 26 may be replaced by another amino
acid, for example, but not limited to threonine. Lysine at position
30 may be replaced by another amino acid, for example, but not
limited to arginine. Other modifications are also possible and are
contemplated herein. Further, the present invention contemplates
variations wherein one or more of the replacements noted above are
present in the polypeptide.
Without wishing to be considered limiting in any manner, and in
respect to SEQ ID NO:2 (GAPDH 2-2-1-1) the alanine residue at
position 18 of SEQ ID NO:2 may be replaced by another amino acid,
for example, but not limited to, proline or serine. The asparagine
residue at position 5 may be replaced with another amino acid, for
example, but not limited to aspartic acid. Other modifications are
also possible and are contemplated herein. Further, the present
invention contemplates polypeptides wherein one or more of the
amino acid replacements noted above are present in the
polypeptide.
Naturally occurring full length GluR2 and GAPDH polypeptides and
the sequences thereof are known in the art. For example, a search
of the National Center for Biotechnology Information using sequence
information provided herein can be used to identify naturally
occurring full length GluR2 and GAPDH protein sequences.
The present invention also provides a polypeptide comprising the
GluR2 NT1-3-2 (Y142-K172) amino acid sequence (SEQ ID NO:1), that
does not encompass a naturally occurring full length GluR2 subunit,
but rather is between about 31 and 200 amino acids in length, for
example, but not limited to 31, 32, 33, 34, 35, 40, 45, 50, 55, 60,
65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170,
180, 190, 200 or any number of amino acids therein between. The
present invention also encompasses polypeptides comprising the
GluR2 NT1-3-2 (Y142-K172) amino acid sequence (SEQ ID NO:1) that
may be defined by a range of lengths of any two of the values
provided above, or any values therein between. For example, but not
to be limiting in any manner, the present invention provides a
polypeptide comprising the GluR2 NT1-3-2 (Y142-K172) amino acid
sequence (SEQ ID NO:1) that is between 31 and 100 amino acids in
length.
The present invention also provides a polypeptide comprising the
GAPDH(2-2-1-1) amino acid sequence (SEQ ID NO:2) that does not
encompass a naturally occurring full length GAPDH protein, but
rather is between about 30 and 334 amino acids in length, for
example, but not limited to 31, 32, 33, 34, 35, 40, 45, 50, 55, 60,
65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170,
180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300,
310, 220, 330, 331, 332, 333, 334, or any number of amino acids
therein between. The present invention also encompasses
polypeptides comprising the GAPDH(2-2-1-1) amino acid sequence (SEQ
ID NO:2) that may be defined by a range of lengths of any two of
the values provided above, or any values therein between. For
example, but not to be limiting in any manner, the present
invention provides a polypeptide comprising the GAPDH(2-2-1-1)
amino acid sequence (SEQ ID NO:2) that is between 31 and 334 amino
acids in length.
The present invention also contemplates polypeptides having an
amino acid sequence that comprises between about 80% to 100%
sequence identity, for example, but not limited to 80%, 81%, 82%,
83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, 99% or 100% identity to the amino acid sequences
described above. Further, the polypeptides may be defined as
comprising a range of sequence identities defined by any two of the
values listed above.
The present invention also contemplates polypeptides that comprise
fragments of GluR2 NT1-3-2 (Y142-K172) amino acid sequence (SEQ ID
NO:1), for example 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19,
18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, and 7 amino acids.
Further, the present invention also contemplates fragments that
exhibit at least about 80% identity, preferably 85%, 90%, 95%, 96%,
97%, 98%, 99% or 100% identity to the polypeptides described above.
The present invention also contemplates polypeptides that comprise
fragments of GAPDH(2-2-1-1), for example 29, 28, 27, 26, 25, 24,
23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, and 7
amino acids. The fragments may comprise N-terminal deletions,
C-terminal deletions, internal deletions or any combination
thereof.
It is also contemplated that the GluR2 NT1-3-2 (Y142-K172) amino
acid sequence (SEQ ID NO:1) or the GAPDH (2-2-1-1) (122'-E250)
amino acid sequence (SEQ ID NO:2) may comprise part of a fusion
protein, for example, but not limited to a polypeptide that further
comprises a heterologous polypeptide or protein, for example, a
carrier protein, a protein transduction domain or the like. For
example, but not wishing to be limiting in any manner, the
polypeptide of the present invention may be fused to a protein
transduction domain to facilitate transit across lipid bilayers or
membranes, for example, but not limited to as described in U.S.
Publication 2002/0142299, U.S. Pat. Nos. 5,804,604, 5,747,641,
5,674,980, 5,670,617, and 5,652,122; PCT publication WO01/15511, US
Publication 2004/0209797, PCT Publication WO99/07728, US
Publication 2003/0186890, all of which are herein incorporated by
reference.
It is also contemplated that the polypeptide of the present
invention may be attached either covalently or non-covalently to a
non-protein substrate or molecule, for example, but not limited to
polyethylene glycol (PEG), dextran or polydextran bead or the like,
a support such as, but not limited to a multi-well plate,
coverslip, array, micro-chip or the like. It is also contemplated
that the polypeptide, non-protein substrate, molecule or any
combination thereof may be labeled, for example with a purification
tag, a radioactive or fluorescent group, enzyme or the like.
The present invention also provides nucleic acids encoding the
polypeptides as described above. In an embodiment of the present
invention which is not meant to be limiting, there is provided a
nucleic acid encoding a polypeptide comprising the GluR2 NT1-3-2
(Y142-K172) amino acid sequence (SEQ ID NO:1) or GAPDH (2-2-2-1)
amino acid sequence (SEQ ID NO:2) that does not encode a naturally
occurring full length GluR2 subunit or GAPDH protein, respectively.
More preferably, but not wishing to be limiting in any manner, the
present invention provides a nucleic acid encoding a polypeptide of
between 31 and 200 amino acids and comprises the GluR2 NT1-3-2
(Y142-K172) amino acid sequence (SEQ ID NO:1) or a polypeptide of
between 30 and 334 amino acids comprising the GAPDH (2-2-1-1) amino
acid sequence (SEQ ID NO:2).
The present invention also contemplates compositions comprising one
or more of the polypeptides and/or nucleic acids of the present
invention. The compositions may comprise one or more diluents,
delivery vehicles, excipients, for example, but not limited to
pharmaceutically acceptable excipients as would be known in the
art, buffers, media, solvents, solutions, carriers or the like.
Such components alone or in any combination may provide a dosage
form for using or administering the polypeptides or nucleic acids
of the present invention to a solution, cell, cell culture, tissue,
organ or subject, for example, but not limited to a human
subject.
To determine whether a nucleic acid exhibits identity with the
sequences presented herein, oligonucleotide alignment algorithms
may be used, for example, but not limited to a BLAST (GenBank URL,
using default parameters: Program: blastn; Database: nr; Expect 10;
filter: default; Alignment: pairwise; Query genetic Codes:
Standard(1)),-BLAST2 (EMBL URL, using default parameters: Matrix
BLOSUM62; Filter: default, echofilter: on, Expect:10, cutoff:
default; Strand: both; Descriptions: 50, Alignments: 50), or FASTA,
search, using default parameters. Polypeptide alignment algorithms
are also available, for example, without limitation, BLAST 2
Sequences (NCBI URL, using default parameters Program: blastp;
Matrix: BLOSUM62; Open gap (11) and extension gap (1) penalties;
gap x_dropoff: 50; Expect 10; Word size: 3; filter: default).
An alternative indication that two nucleic acid sequences are
substantially identical is that the two sequences hybridize to each
other under moderately stringent, or preferably stringent,
conditions. Hybridization to filter-bound sequences under
moderately stringent conditions may, for example, be performed in
0.5 M NaHPO.sub.4, 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at
65.degree. C., and washing in 0.2.times.SSC/0.1% SDS at 42.degree.
C. for at least 1 hour (see Ausubel, et al. (eds), 1989, Current
Protocols in Molecular Biology, Vol. 1, Green Publishing
Associates, Inc., and John Wiley & Sons, Inc., New York, at p.
2.10.3). Alternatively, hybridization to filter-bound sequences
under stringent conditions may, for example, be performed in 0.5 M
NaHPO.sub.4, 7% SDS, 1 mM EDTA at 65.degree. C., and washing in
0.1.times.SSC/0.1% SDS at 68.degree. C. for at least 1 hour (see
Ausubel, et al. (eds), 1989, supra). Hybridization conditions may
be modified in accordance with known methods depending on the
sequence of interest (see Tijssen, 1993, Laboratory Techniques in
Biochemistry and Molecular Biology--Hybridization with Nucleic Acid
Probes, Part I, Chapter 2 "Overview of principles of hybridization
and the strategy of nucleic acid probe assays", Elsevier, New
York). Generally, but not wishing to be limiting, stringent
conditions are selected to be about 5.degree. C. lower than the
thermal melting point for the specific sequence at a defined ionic
strength and pH.
A polypeptide of the invention can be synthesized in vitro or
delivered to a cell in vivo by any conventional method. As a
representative example of an in vitro method, the polypeptide may
be chemically synthesized in vitro, or may be enzymatically
synthesized in vitro in a suitable biological expression system. As
a representative example of an in vivo method, a DNA, RNA, or
DNA/RNA hybrid molecule comprising a nucleotide sequence encoding a
polypeptide of the invention is introduced into an animal, and the
nucleotide sequence is expressed within a cell of an animal.
The nucleotide sequence may be operably linked to regulatory
elements in order to achieve preferential expression at desired
times or in desired cell or tissue types. Furthermore, as will be
known to one of skill in the art, other nucleotide sequences
including, without limitation, 5' untranslated region, 3'
untranslated regions, cap structure, poly A tail, translational
initiators, sequences encoding signalling or targeting peptides,
translational enhancers, transcriptional enhancers, translational
terminators, transcriptional terminators, transcriptional
promoters, may be operably linked with the nucleotide sequence
encoding a polypeptide (see as a representative example "Genes
VII", Lewin, B. Oxford University Press (2000) or "Molecular
Cloning: A Laboratory Manual", Sambrook et al., Cold Spring Harbor
Laboratory, 3rd edition (2001)). A nucleotide sequence encoding a
polypeptide or a fusion polypeptide comprising the polypeptide may
be incorporated into a suitable vector. Vectors may be commercialy
obtained from companies such as Stratagene or InVitrogen. Vectors
can also be individually constructed or modified using standard
molecular biology techniques, as outlined, for example, in Sambrook
et al. (Cold Spring Harbor Laboratory, 3rd edition (2001)). A
vector may contain any number of nucleotide sequences encoding
desired elements that may be operably linked to a nucleotide
sequence encoding a polypeptide or fusion polypeptide comprising a
protein transduction domain. Such nucleotide sequences encoding
desired elements, include, but are not limited to, transcriptional
promoters, transcriptional enhancers, transcriptional terminators,
translational initiators, translational terminators, ribosome
binding sites, 5' untranslated region, 3' untranslated regions, cap
structure, poly A tail, origin of replication, detectable markers,
afffinity tags, signal or target peptide, Persons skilled in the
art will recognize that the selection and/or construction of a
suitable vector may depend upon several factors, including, without
limitation, the size of the nucleic acid to be incorporated into
the vector, the type of transcriptional and translational control
elements desired, the level of expression desired, copy number
desired, whether chromosomal integration is desired, the type of
selection process that is desired, or the host cell or the host
range that is intended to be transformed.
As described herein, and unless clearly indicated otherwise, the
term "mini-gene" means the expression product of a nucleic acid or
nucleotide sequence encoding and capable of expressing a
polypeptide in a cell. For example, but not wishing to be
considered limiting in any manner, a mini-gene includes a nucleic
acid or nucleotide sequence encoding and capable of expressing the
polypeptide comprising the GluR2 NT1-3-2 (Y142-K172) amino acid
sequence (SEQ ID NO:1) in a cell. In an alternate embodiment, the
mini-gene comprises a nucleic acid or nucleotide sequence encoding
and capable of expressing the polypeptide comprising the
GAPDH(2-2-1-1) (I221-E250) amino acid sequence (SEQ ID NO:2) in a
cell.
The DNA, RNA, or DNA/RNA hybrid molecule may be introduced
intracellularly, extracellularly into a cavity, interstitial space,
into the circulation of an organism, orally, or by any other
standard route of introduction for therapeutic molecules and/or
pharmaceutical compositions. Standard physical methods of
introducing nucleic acids include, but are not limited to,
injection of a solution comprising RNA, DNA, or RNA/DNA hybrids,
bombardment by particles covered by the nucleic acid, bathing a
cell or organism in a solution of the nucleic acid, or
electroporation of cell membranes in the presence of the nucleic
acid.
A nucleic acid may be introduced into suitable eukaryotic cells ex
vivo and the cells harbouring the nucleic acid can then be inserted
into a desired location in an animal. A nucleic acid can also be
used to transform prokaryotic cells, and the transformed
prokaryotic cells can be introduced into an animal, for example,
through an oral route. Those skilled in the art will recognize that
a nucleic acid may be constructed in such a fashion that the
transformed prokaryotic cells can express and secrete a polypeptide
of the invention. Further, a nucleic acid may also be inserted into
a viral vector and packaged into viral particles for efficient
delivery and expression.
Dosage Forms
The polypeptides of the present invention or the nucleic acids
encoding the polypeptides of the present invention may be
formulated into any convenient dosage form as would be known in the
art. The dosage form may comprise, but is not limited to an oral
dosage form wherein the agent is dissolved, suspended or the like
in a suitable excipient such as but not limited to water or saline.
In addition, the agent may be formulated into a dosage form that
could be applied topically or could be administered by inhaler, or
by injection either subcutaneously, into organs, or into
circulation. An injectable dosage form may include other carriers
that may function to enhance the activity of the agent. Any
suitable carrier known in the art may be used. Also, the agent may
be formulated for use in the production of a medicament. Many
methods for the productions of dosage forms, medicaments, or
pharmaceutical compositions are well known in the art and can be
readily applied to the present invention by persons skilled in the
art.
According to the present invention there is also provided a method
of inhibiting GluR2 subunit association with GAPDH comprising:
administering a polypeptide comprising the GluR2 NT1-3-2
(Y142-K172) amino acid sequence (SEQ ID NO:1) to a solution, cell,
cell culture, tissue or subject comprising GluR2 subunit and GAPDH.
The method may be practiced in vitro or in vivo. In an embodiment
wherein the method is practiced in vivo, the method may be
practiced in a human subject. The human subject may have or be
susceptible to stroke, epilepsy or other forms of brain injury.
The invention also provides a method of inhibiting GluR2 subunit
association with GAPDH comprising: administering a nucleic acid
capable of expressing a polypeptide comprising the GluR2 NT1-3-2
(Y142-K172) amino acid sequence (SEQ ID NO:1) to a cell, cell
culture, tissue or subject comprising GluR2 subunit and GAPDH. In
an embodiment wherein the method is practiced in vivo, the method
may be practiced in a human subject. The human subject may have or
be susceptible to stroke, epilepsy or other forms of brain injury,
for example, but not limited to traumatic brain injury or injury
from cardiac bypass surgery.
According to the present invention there is also provided a method
of inhibiting GAPDH association with p53 comprising: administering
a polypeptide comprising the GAPDH (2-2-1-1) amino acid sequence
(SEQ ID NO:2) to a solution, cell, cell culture, tissue or subject
comprising GAPDH and p53. The method may be practiced in vitro or
in vivo. In an embodiment wherein the method is practiced in vivo,
the method may be practiced in a human subject. The human subject
may have or be susceptible to stroke, epilepsy or other forms of
brain injury.
The invention also provides a method of inhibiting GAPDH
association with p53 comprising: administering a nucleic acid
capable of expressing a polypeptide comprising the GAPDH (2-2-1-1)
amino acid sequence (SEQ ID NO:2) to a cell, cell culture, tissue
or subject comprising GAPDH and p53. In an embodiment wherein the
method is practiced in vivo, the method may be practiced in a human
subject. The human subject may have or be susceptible to stroke,
epilepsy or other forms of brain injury.
Also provided by the present invention is a method of inhibiting
AMPA receptor mediated excitotoxicity comprising,
Administering, a polypeptide comprising the GluR2 NT1-3-2
(Y142-K172) amino acid sequence (SEQ ID NO:1) or a polypeptide
comprising the GAPDH (2-2-1-1) amino acid sequence (SEQ ID NO:2);
or a nucleic acid capable of expressing a polypeptide comprising
the GluR2 NT1-3-2 (Y142-K172) amino acid sequence (SEQ ID NO:1), or
a nucleic acid capable of expressing a polypeptide comprising the
GAPDH (2-2-1-1) amino acid sequence (SEQ ID NO:2) to a cell, tissue
or subject in need thereof. Accordingly, the method may be
practiced in vitro or in vivo. In respect of a method that is
practiced in vivo, but without wishing to be limiting in any
manner, the subject may have or be at risk of stroke, epilepsy, or
other forms of brain injury.
In still a further embodiment of the present invention, which is
not meant to be limiting in any manner, there is provided a method
of treating or preventing brain injury comprising, administering a
polypeptide comprising the GluR2 NT1-3-2 (Y142-K172) amino acid
sequence (SEQ ID NO:1) or a polypeptide comprising the GAPDH
(2-2-1-1) amino acid sequence (SEQ ID NO:2); or a nucleic acid
capable of expressing a polypeptide comprising the GluR2 NT1-3-2
(Y142-K172) amino acid sequence (SEQ ID NO:1), or a nucleic acid
capable of expressing a polypeptide comprising the GAPDH (2-2-1-1)
amino acid sequence (SEQ ID NO:2), to a subject in need thereof. As
will be evident to a person of skill in the art, an embodiment that
comprises administering a nucleic acid as described above, further
comprises the step of expressing nucleic acid in the subject.
The present invention also contemplates a method as defined above
wherein the polypeptide is administered prior to, during, after or
both prior to and after an event that causes or may cause brain
injury, for example, but not limited to stroke, epileptic seizure,
brain damage resulting from cardiac bypass surgery or a combination
thereof. For example, but not to be considered limiting in any
manner, subjects diagnosed with epilepsy may be administered the
polypeptide of the present invention at one or more intervals after
being diagnosed with the condition, preferably prior to, during or
after prolonged episodes of seizure.
In a preferred embodiment, the polypeptide or polypeptides of the
present invention are administered immediately after, for example,
but not limited to, about concurrently with an event that causes,
or is capable of causing brain injury and about 24 hours
thereafter, more preferably about 12 hours, still more preferably
about 6 hours, still more preferably about 2 hours, more preferably
1 hour or less.
Also provided by the present invention is a kit that comprises: a)
a polypeptide comprising the GluR2 NT1-3-2 (Y142-K172) amino acid
sequence (SEQ ID NO:1), b) a nucleic acid capable of expressing a
polypeptide comprising the GluR2 NT1-3-2 (Y142-K172) amino acid
sequence (SEQ ID NO:1), c) a polypeptide that comprises GAPDH
(2-2-1-1) amino acid sequence (SEQ ID NO:2), d) a nucleic acid
capable of expressing a polypeptide comprising the GAPDH (2-2-1-1)
amino acid sequence (SEQ ID NO:2) e) one or more diluents, delivery
vehicles, pharmaceutically acceptable excipients or a combination
thereof, 0 one or more devices for delivering polypeptides or
nucleic acids to a solution, cell, cell culture, tissue, organ or
subject, g) instructions for using any component in the kit or
practicing any method as described herein, or any combination
thereof. Further, the kit may comprise other components as would be
known to a person of skill in the art.
The present invention will be further illustrated in the following
examples.
EXAMPLES
Experimental Procedures
Primary Hippocampal Neuron Culture
Primary cultures from hippocampus were prepared from fetal Wistar
rats (embryonic day 17-19) on Cell.sup.+ (Sarstedt) culture dishes
as previously described (73). The cultures were used for
experiments on 12-15 d after plating. Hippocampal cultures were
pretreated withGluR2 NT1-3-2 peptides before kainic acid
treatment.
GST Fusion Proteins and Mini-Genes
To construct GST-fusion proteins and mini-genes expressing
truncated GluR2.sub.NT and GAPDH, cDNA fragments were amplified by
using PCR method with specific primers. Except where specified, all
5' and 3' oligonucleotides incorporated BamH1 site (GGATCC) and
XhoI sites (CTCGAG), respectively, to facilitate subcloning into
vector pcDNA3 (for mini-gene construction) or into vector pGEX-4T3
(for GST-fusion protein construction). GST-fusion proteins were
prepared from bacterial lysates as described by the manufacturer
(Amersham). To confirm appropriate splice fusion and the absence of
spurious PCR generated nucleotide errors, all constructs were
resequenced.
Protein Affinity Purification, In Vitro Binding,
Co-Immunoprecipitation and Western Blot
Protein affinity purification, in vitro binding,
co-immunoprecipitation and Western blot analyses were performed as
previously described (73, 79). Antibodies used for
immunoprecipitation, Western blots and cell surface ELISA assays
included GAPDH (polyclonal from Abcam, monoclonal from Chemicon),
GluR2 (Western blots: Chemicon; immunoprecipitation: Upstate), HA
(monoclonal, Covance), .alpha.-tubulin (monoclonal, Sigma-Aldrich),
LaminB1 (Zymed Laboratories).
Cell-ELISA Assays
Cell-ELISA assays (colorimetric assays) were done essentially as
previously described (82). In brief the same density of HEK-293T
cells transfected with cDNAs encoding various receptor constructs
were treated with 100 .mu.M glutamate or extracellular solution
(ECS) before fixing in 4% (W/V) paraformaldehyde for 10 minutes in
the absence (non-permeabilized conditions) or presence
(permeabilized conditions) of 1% (V/V) Triton X-100. Cells were
incubated in 1% (W/V) glycine for 10 minutes at 4.degree. C. to
recover from the fixing. Cells were then incubated with a
monoclonal antibody against specific antibodies for the purpose of
labeling the receptors or proteins on the cell surface under
non-permeabilized conditions or the entire receptor pool under
permeabilized conditions. After incubation with corresponding
HRP-conjugated secondary antibodies (Sigma-Aldrich), the HRP
substrate o-phenylenediamine (Sigma-Aldrich Co) was added to
produce a color reaction that was stopped with the equal volume of
3N HCl. Fluorescence intensity in each well was measured with a
plate reader (Victor3; PerkinElmer). The cell surface expression of
HA-GluR2 after pre-treatment with glutamate was presented as the
ratio of colorimetric readings under non-permeabilized conditions
to those under permeabilized conditions, and then normalized to
their respective control groups (pretreated with ECS). Afterwards,
cells were scrapped from the dishes, and the protein concentration
of each dish was measured. The results of cell surface expression
of receptors or proteins were calibrated by the protein
concentration of each well. Analysis was done using at least 9
separate wells in each group. Cell ELISA using primary hippocampal
neurons was performed identically with assays using HEK-293T cells,
with the exception that the anti-GluR2 antibody (MAB397; Chemicon)
was used as primary antibody instead of anti-HA.
Quantification of AMPA-Mediated Excitotoxicity
An equal density of HEK-293T cells transfected with AMPAR was
exposed to 300 .mu.M glutamate/25 .mu.M cyclothiazide at 37.degree.
C. for 24 hour. Cells were allowed to recover for 24 hours at
37.degree. C. To quantify AMPA-mediated cell death, culture medium
was replaced by extracellular solution containing 50 .mu.g/ml of
propidium iodide (PI) (Invitrogen). After 30 minutes incubation at
37.degree. C., fluorescence intensity in each well was measured
with a plate reader (Victor3; PerkinElmer). The fraction of dead
cells was normalized to the cell toxicity that occurred in either
the glutamate treated cells or KA treated neurons. Primary
hippocampal neurons were exposed to 100 .mu.M KA/25 .mu.M
cyclothiazide in medium at 37.degree. C. for 1 hour, at 37.degree.
C.
Cell Biotinylation
Cell biotinylation was essentially performed as described
previously (76, 83). Briefly, for cell surface biotinylation, cells
were rinsed four times with ice-cold PBS containing 0.1 mM
CaCl.sub.2 and 1.0 mM MgCl.sub.2 (PBS.sup.2+) after treatment, and
incubated twice with 1.0 mg/ml sulfo-NHS-LC-biotin (Pierce,
Rockford, Ill.) for 20 minutes at 4 degree. Non-reactive biotin was
quenched with twice with 20 minute's incubation at 4 degree in
ice-cold PBS.sup.2+ and 0.1 M glycine. Cells were solubilized in
RIPA buffer (10 mM Tris, Ph7.4, 150 mM NaCl, 1.0 mM EDTA, 0.1%
(W/V) SDS, 1.0% (V/V) Trition X-100 and 1.0% (V/V) Sodium
deoxycholate) containing protease inhibitors (1.0 mM PMSF and 1.0
.mu.g/ml protease cocktail). Biotinylated and non-biotinylated
proteins were separated from equal amounts of cellular protein by
incubation with 50 .mu.l of 50% slurry of immobilized
streptavidin-conjugated beads (Pierce, Rockford, Ill.) for
overnight with constant mixing at 4 degree. Unbound proteins
(supernatant) were saved for later co-immunoprecipitation
experiment. Proteins bound to streptavidin beads were eluted in
biotin elution buffer. Biotinylated and non-biotinylated samples
were applied to protein A/G PLUS-agarose (Santa Cruz) for
co-immunoprecipitation. For nuclear biotinylated proteins, cells
were firstly incubated with 1.0 mg/ml sulfo-NHS-SS-biotin (Pierce,
Rockford, Ill.) before treatment. Afterwards cells were treated
with 50 mM glutathione to cleave all cell surface biotin and nuclei
were extracted from cell lysates. After incubation with immunopure
immobilized streptavidin-conjugated beads (Pierce, Rockford, Ill.),
beads were washed four times with RIPA buffer. The bead pellets
were boiled in sample buffer and subjected to Western blot
analysis.
Purification of Nuclei
Nuclei isolation was prepared as described previously (55, 66).
Briefly, cells were gently rinsed twice with ice-cold PBS. And
scraped in 1 ml of solution 1 (10 mM Tris-HCl, pH7.4, 100 mM NaCl2,
2.5 mM MgCl2, 0.5% NP-40, proteinase inhibitor and PMSF) per 10-cm
plate. Then cells were homogenized by four passages through a
25-gauge needle and spin at 3000 g briefly. Pellets containing
nuclei were subsequently utilized in biochemical assays.
Example 1
GAPDH Interacts with the Amino-Terminus of the GluR2 Subunit
To identify proteins that might possibly interact with N-terminus
(NT) of AMPA receptor GluR1 and GluR2 subunits, we incubated rat
hippocampal extracts with GST-fusion proteins: GST-GluR1NT
(A19-E538), GST-GluR2NT (V22-E545), and GST alone, respectively.
Then samples were subjected onto 10% SDS-PAGE and stained with
Coomassie blue 8250. A single immunoreactive band with an apparent
molecular mass of .about.37 kDa was enriched in GST-GluR2NT
precipitated sample but not in that of GST-GluR1 NT or GST alone.
We excised the .about.37 kDa band from the gel and used mass
spectrometry to identify the protein. The most significant score
for this band was obtained with GAPDH (Table 1).
TABLE-US-00001 TABLE 1 Protein Analysis Results Database: NCBInr
(2314886 sequences; 787107140 residues) Taxonomy: Mammalia
(mammals) (340771 sequences) Peptide Protein AC Mass Score Matched
Taxonomy Glyceraldehyde 3- gi|56188 36103 128 3 Rattus phosphate-
norvegicus dehydrogenase Matched Peptides Mr(expt) Mr(calc) Score
peptide 2245.13 2244.09 15 VIISAPSADAPMFVMGVNHEK (SEQ ID NO: 6)
2611.92 2610.35 63 VIHDNFGIVEGLMTTVHAITATQK (SEQ ID NO: 7) 1557.75
1556.79 50 VPTPNVSVVDLTCR (SEQ ID NO: 8) Sequence Coverage: 17% 1
MVKVGVNGFG RIGRLVTRAA FSCDKVDIVA INDPFIDLNY MVYMFQYDST 51
HGKFNGTVKA ENGKLVINGK PITIFQERDP VKIKWGDAGA EYVVESTGVF 101
TTMEKAGAHL KGGAKRVIIS APSADAPMFV MGVNHEKYDN SLKIVSNASC 151
TTNCLAPLAK VIHDNFGIVE GLMTTVHAIT ATQKTVDGPS GKLWRDGRGA 201
AQNIIPASTG AAKAVGKVIP ELNGKLTGMA FRVPTPNVSV VDLTCRLEKP 251
AKYDDIKKVV KQAAEGPLKG ILGYTEDQVV SCDFNSNSHS STFDAGAGIA 301
LNDNIVKLIS WYDNEYGYSN RVVDLMAYMA SKE (SEQ ID NO: 9)
Example 2
Identification of Interaction Sites of the GAPDH and the GluR2
Subunit Complex
In order to delineate the region of the GluR2 NT involved in the
interaction with the GAPDH, three GluR2 NT GST-fusion proteins
[GluR2 NTa V22-S271, (250 a.a), GluR2 NTb K272-421, (150 a.a),
GluR2 NTc L422-E545, (124 a.a)] were constructed (FIG. 4A). In
affinity purification assays, GluR2 NTa, but not GluR2 NTb, GluR2
NTc or GST alone precipitated GAPDH in rat hippocampal brain
extract (FIG. 4B). Although these results demonstrated the presence
of a GAPDH and GluR2 NT complex, it could not determine whether the
complex was formed through a direct or indirect interaction. To
clarify the nature of the interaction, blot overlay experiments
were performed, which provided in vitro evidence for a direct
interaction. GluR2 NTa, GluR2 NTb and GluR2 NTc were probed with in
vitro translated [35S]-methionine labelled peptides encoding GAPDH
([35S]-GAPDH). The [35S]-GAPDH probe bound with GluR2NTa, but not
GluR2NTb or GluR2NTc. The binding of [35S]-GAPDH was specific, as
it did not bind with GST (FIG. 4C).
In order to further delineate the region of GluR2 NTa involved in
the interaction with GAPDH, the GluR2 NTa region was further
divided into five GST-fusion proteins that were composed of 50
amino acids each (NTa1: V22-F71, GluR2 NTa2: A72-T121, GluR2 NTa3:
H122-K171, GluR2 NTa4: K172-D221, GluR2 NTa5: Q222-S271)(FIG. 5A).
In affinity purification assays, GluR2 NTa3, but not the other
sub-regions or GST alone, precipitated GAPDH in rat hippocampal
extracts (FIG. 5B). This was supported by a blot overlay experiment
(FIG. 5C). Here the [35S]-GAPDH probe bound with GluR2 NTa3, but
not to any of the other constructs.
We then further divided the GluR2 NTa3 region into 30 amino acids
GST-fusion proteins and mini-genes (GluR2 NTa3-1: H122-E141, 20
a.a; GluR2 NT1-3-2 Y142-K172, 30 a.a) to delineate the region of
GluR2 NTa3 involved in the interactions (FIG. 6A). GluR2 NT1-3-2,
but not GluR2 NTa3-1 or GST alone, precipitated GAPDH in rat
hippocampal extracts in affinity purification assays (FIG. 6B).
This was also supported by a blot overlay experiment where
[35S]-GAPDH probe bound with GluR2 NT1-3-2, but not GluR2 NTa3-1 or
GST alone (FIG. 6C).
In order to confirm the existence of GAPDH and GluR2NT complexes,
we examined if GAPDH could CO-IP with GluR2 subunit in rat
hippocampal extracts. The GAPDH antibody precipitated GluR2 subunit
suggesting a physical interaction between GAPDH and GluR2 subunit
(FIG. 3A). Next we performed the protein affinity purification
assays to further confirm whether the N-terminus or the C-terminus
of GluR2 subunit was involved in the formation of complex.
GST-GluR2NT, but not GST-GluR2CT or GST alone, precipitated GAPDH
(FIG. 3B).
GAPDH and AMPAR Form a Direct Protein-Protein Through the GluR2
Amino-Terminus
In an attempt to validate potential protein regions that interact
with the GluR2 subunit, we repeated experiments using
GST-GluR2.sub.NT (V.sub.22-E.sub.545) to affinity "pull down"
proteins from solubilized rat hippocampal tissues, using GST alone
and GST-GluR1.sub.NT (A.sub.19-E.sub.538) as controls. The
precipitated proteins were then identified by Coomassie brilliant
blue staining after SDS-PAGE. A prominent protein band of -40 kD
was specifically precipitated by GST-GluR2.sub.NT, but not by GST
alone or GST-GluR1.sub.NT (FIG. 6D). These results suggested that
the GluR2 subunit may form a protein complex with GAPDH through the
GluR2.sub.NT. We then confirmed this GluR2.sub.NT-GAPDH putative
interaction through pull-down/affinity purification experiments
using GST-GluR2.sub.NT, GST-GluR2.sub.CT (I.sub.833-I.sub.883) and
GST alone. Subsequent Western blot analysis using a GAPDH antibody
confirmed an association between GluR2.sub.NT and GAPDH (FIG.
6E).
In order to confirm previous results and to delineate the region(s)
of the GluR2.sub.NT involved in the interaction with GAPDH, three
GluR2.sub.NT GST-fusion proteins (GluR2.sub.NT1:
V.sub.22-S.sub.271, GluR2.sub.NT2: K.sub.272-I.sub.421,
GluR2.sub.NT3: L.sub.422-E.sub.545) were constructed (FIG. 6F) and
utilized in affinity purification assays. As shown in FIG. 6J, only
GST-GluR2.sub.NT1 precipitated GAPDH indicating that the GluR2
subunit could interact with GAPDH through its NT region
V.sub.22-S.sub.271. We then created a series of truncations of the
GluR2.sub.NT1 region to map the site that interacted with GAPDH. As
shown in FIGS. 6K and 6L, only GST-GluR2.sub.NT1-3
(H.sub.122-K.sub.172) and GST-GluR2NT1-3-2 (Y.sub.142-K.sub.172)
were able to precipitate GAPDH from rat hippocampal tissue. While
these results demonstrated the presence of the GAPDH: AMPAR protein
complex in rat hippocampal tissue, they did not clarify whether the
GAPDH: AMPAR protein complex was formed through a direct
interaction between GAPDH and AMPAR or was an indirect interaction
facilitated by an accessory binding protein. In vitro binding
assays provided evidence that GAPDH and the GluR2 subunit could
directly interact with each other. As shown in FIG. 6, in vitro
translated [.sup.35S]-GAPDH probe hybridized with GST-GluR2.sub.NT1
but not GST-GluR2.sub.NT2, GST-GluR2.sub.NT3 or GST alone,
indicating the specificity of the direct protein-protein
interaction between GAPDH and GluR2.sub.NT1. Consistent with our
affinity purification experiments, in vitro translated
[.sup.35S]-GAPDH probe only hybridized with GST-GluR2.sub.NT1-3 and
GST-GluR2 NT1-3-2, (FIGS. 6K, L). These data suggested that GAPDH
was involved in a direct protein-protein interaction with the GluR2
subunit through the Y 142-K172 region of the GluR2.sub.NT.
Example 3
Agonist Regulation of GluR2NT-GAPDH Protein-Protein
Interactions
Before investigating whether the direct protein-protein interaction
between GAPDH and GluR2 NT have functional implications, we tested
if AMPA receptor activation affected the observed interactions.
Based on previous reports, we focused on the GluR1/GluR2 AMPA
receptor combination, one of the two most common AMPA receptor
subunit combinations in the hippocampus, which have important
defined roles in AMPA receptor trafficking and synaptic
plasticity.
We co-expressed both GluR1 and GluR2 along in the presence or
absence of the GluR2 NT1-3-2 mini-gene in HEK293T cells. It should
be noted that HEK293T cells expresses endogenous GAPDH. The GluR2
subunit and GAPDH could associate without exogenous AMPA receptor
agonist stimulation (FIG. 7). The insertion of mini-gene greatly
interrupted the protein-protein interaction.
Activation of AMPA receptor with the agonist glutamate resulted in
an increase in the CO-IP of GAPDH by the GluR2 subunit antibody
(FIG. 8). The association between GAPDH and GluR2 was decreased by
the application of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), a
competitive AMPA receptor antagonist used to block the AMPA
receptor activation (FIG. 8). While the insertion of the GluR2
NT1-3-2 mini-gene was able to interrupt the protein-protein
interaction either with or without glutamate treatment (FIGS. 7,
9). In all immunoprecipitation experiments, the level of directly
immunoprecipitated GluR2 subunit was used as a loading control
(FIG. 7-9). Glutamate stimulation did not significantly alter the
initial levels of solubilized protein GluR2 subunit (FIG. 10) or
GAPDH (FIG. 11).
Example 4
Translocation of the GluR2 Subunit and GAPDH
The association of the GluR2 subunit with GAPDH increased after the
glutamate treatment, and the expression level of GAPDH and GluR2
subunit changed little in the whole cell protein, accordingly, the
expression level of these proteins in different cell compartments
was tested.
We extracted different cell compartments of transfected HEK 293T
cells, such as cytosol, nucleus and mitochondria. Glutamate
treatment (100 mM) facilitated GAPDH translocation from the cytosol
and mitochondria to the nucleus. The insertion of the GluR2 NT1-3-2
mini-gene was able to interrupt the GAPDH translocation triggered
by glutamate treatment (FIG. 12). The expression level of the GluR2
subunit increased in the nucleus after the glutamate treatment,
while the insertion of the GluR2 NT1-3-2 mini-gene also diminished
this increase (FIG. 12).
We also examined the translocation of GAPDH and the GluR2 subunit
in hippocampal neuron cultures. Because neurons have other
glutamate receptors such as the NMDA receptor, we used the AMPA
receptor-selective agonist KA (100 .mu.M) other than glutamate. The
TAT-GluR2 NT1-3-2 peptide (10 .mu.M) was applied for 30 minutes
before KA treatment. The expression level of the GluR2 subunit
increased in the nucleus after KA treatment, while intracellular
application of GluR2 NT1-3-2 peptide reversed this increase (FIG.
12).
Example 5
Functional Characterization of the GAPDH and GluR2
Interaction-Modulation of GluR2 Cell Surface Expression Through the
GAPDH and GluR2 Interaction
Modification of ligand-gated receptor function at the postsynaptic
domain is one of the most important mechanisms by which the
efficacy of synaptic transmission in the nervous system is
regulated. Traditionally, these types of modifications have been
achieved mainly by altering the channel-gating properties or
conductance of the receptors. However, recent evidence suggests
that AMPA receptors are continuously recycled between the plasma
membrane and the intracellular compartments via vesicle-mediated
plasma membrane insertion and clathrin-dependent endocytosis.
Regulation of either receptor insertion or endocytosis results a
rapid change in the population of these receptors expressed on the
plasma membrane surface and in the receptor-mediated responses.
Therefore, the regulation plays an important role in mediating
certain forms of synaptic plasticity. In order to investigate
whether the population of AMPA receptors on the plasma membrane can
be regulated by the GAPDH and GluR2 NT complex, we scanned
transiently transfected HEK293T cells and hippocampal neurons
expressing AMPA receptors by cell ELISA. The oxygen-glucose
deprivation (OGD) model was also applied.
In HEK293T cells coexpressing both GluR1 and GluR2 subunits of AMPA
receptors, the insertion of GluR2 NT1-3-2 mini-gene did not change
the number of GluR2 subunit on plasma membrane (FIG. 13). However,
after treatment with glutamate (100 .mu.M) for 30 minutes, the
plasma membrane expression of GluR2 subunits significantly
increased in GluR2 NT1-3-2 insertion group, compared to the
mini-gene sham-transfected group (FIG. 13).
We also examined GluR2 subunit expression at the plasma membrane in
hippocampal neuron culture. The TAT-GluR2 NT1-3-2 peptide (10
.mu.M) was applied for 30 minutes before KA treatment.
Intracellular application of GluR2 NT1-3-2 peptide significantly
increased the cell surface expression of GluR2 subunits after KA
treatment (FIG. 14).
We further tested GluR2 subunit expression at the plasma membrane
in the OGD model. Hippocampal neurons were deprived of oxygen and
glucose for 2 hours and allowed to recover for 24 hours. The
TAT-GluR2 NT1-3-2 peptide (10 .mu.M) was applied for 30 minutes OGD
treatment. Intracellular application of GluR2 NT1-3-2 peptide also
increased the cell surface expression of GluR2 subunits in the OGD
model (FIG. 15).
Altogether, these data suggest that the GAPDH and GluR2 association
plays an important role in the trafficking of AMPA receptors, which
may in turn affect synaptic plasticity.
Example 6
Modulation of the AMPA Receptor-Mediated Excitotoxicity Through the
GAPDH and GluR2 Interaction
Although the protein-protein interaction between GAPDH and GluR2 NT
might play an important role in the trafficking of AMPA receptors,
it is still unclear whether the interaction is responsible for the
observed AMPA-mediated cell death. To further investigate the
functional implication of this biochemical interaction between
GAPDH and GluR2 NT, we tested the effects of this interaction on
AMPA receptor-mediated excitotoxicity in both transfected HEK293T
cells and hippocampal culture neurons. The OGD model was also
applied.
The AMPA receptor-mediated excitotoxicity was induced by the
incubation with 100 mM glutamate. HEK293T cells were transfected
with GluR1 and GluR2 subunits alone or in the presence or absence
of the GluR2 NT1-3-2 mini-gene. We quantified the AMPA-mediated
excitotoxicity by using a PI fluorescence assay. To define the
effect of the observed interaction, we examined whether the
blockade of the GAPDH and GluR2 NT interaction by using GluR2
NT1-3-2 mini-gene would affect the AMPA-mediated exciotoxicity.
With the 100 mM glutamate treatment, the AMPA receptor-mediated
cell death was greatly reduced by the overexpression of GluR2
NT1-3-2 mini-gene, compared to mini-gene sham-transfected group
(FIG. 16).
We also examined the AMPA receptor-mediated excitotoxicity in
hippocampal culture neurons. Hippocampal neurons were pretreated
with either 10 .mu.M TAT only or the TAT-GluR2 NT1-3-2 peptide for
30 minutes. The excitotoxicity was induced by incubation with 100
.mu.M KA and 30 .mu.M cyclothiazide (to prevent AMPA receptor
desensitization). The neurons were allowed to recover for 24 hours.
In the TAT-GluR2 NT1-3-2 group, AMPA receptor-mediated
excitotoxicity was reduced when compared to the TAT only group
(FIG. 17).
We further tested the AMPA receptor-mediated excitotoxicity in the
OGD model. Hippocampal neurons were pretreated with either 10 .mu.M
TAT only or the TAT-GluR2 NT1-3-2 peptide for 30 minutes. The
excitotoxicity was induced by incubation with the OGD treatment.
The neurons were allowed to recover for 24 hours. In the TAT-GluR2
NT1-3-2 group, AMPA receptor-mediated excitotoxicity was reduced
when compared to the TAT only group in OGD treatment (FIG. 18).
These data from the HEK 293T cells, hippocampal neurons and OGD
models strongly suggest that the protein-protein interaction
between the GAPDH and GluR2 NT is essential for the AMPA
receptor-mediated excitotoxicity.
Example 7
Potential Molecules Involved in the Regulation of the Function of
The GluR2 Subunit and GAPDH Complex and their Translocation
In order to determine the potential molecules involved in the
regulation of the AMPA receptor-mediated excitotoxicity, we focused
on several molecules in the cell death pathway, such as poly
ADP-ribose polymerase (PARP), P53, caspase-3, Bcl-2 and Bcl-x. In
transfected HEK 293T cells, glutamate treatment (100 .mu.M) and the
insertion of the GluR2 NT1-3-2 mini-gene affected the expression
level of PARP, caspase-3, Bc1-2 and Bcl-x (FIG. 19).
There is no nuclear localization signal on GAPDH, while there are
some potential nuclear localization signals in the amino terminus
of the GluR2 subunit. We also tested the potential protein which
might lead GAPDH translocation from other cell compartments to the
nucleus. Apoptosis inducing factor (AIF) possesses both
mitochondria and nuclear localization signals. In the affinity
purification assay, GST-fusion protein GST-GluR2 NT and GST-GAPDH,
but not GST-GluR2CT or GST alone, precipitated AIF in rat
hippocampal extracts. Altogether, these data suggest that several
molecules are involved in the regulation of the trafficking of AMPA
receptors and GAPDH, as well as AMPA-receptor mediated
excitotoxicity.
Example 8
Agonist Regulation of Extracellular GAPDH:AMPAR Complex
Formation
To investigate whether GAPDH forms a complex with AMPAR in vivo, we
performed to co-immunoprecipitation experiments with proteins
extracted from the rat hippocampus. As shown in FIG. 21A,
immunoprecipitation of GluR2 was able to co-precipitate GAPDH from
solubilized proteins extracted from rat hippocampus, indicating a
physical interaction between GluR2 and GAPDH may occur in vivo. We
then tested if AMPAR activation affected the observed GAPDH-GluR2
interaction. Although GAPDH and AMPAR could associate with each
other without exogenous stimulation in HEK-293T cells expressing
GluR1/GluR2 subunits (FIG. 21B, top panel) and in primary cultures
of rat hippocampal neurons (FIG. 21C, top panel), activation of
AMPAR resulted in a 75.+-.18% (mean.+-.SE, n=3) and 58.+-.11%
(mean.+-.SE, n=3) increase in the co-immunoprecipitation of GAPDH
with GluR2, respectively. Agonist stimulation did not significantly
alter the levels of directly immunoprecipitated GluR2 subunit
(FIGS. 21B, C, bottom panels). Interestingly, preincubation of the
GluR2 NT1-3-2 peptide (10 .mu.M, 1 hour) significantly inhibited
the agonist-induced increase in the GAPDH: AMPAR complex formation
in HEK-293T cells expressing GluR1/GluR2 (FIG. 21B) and in
hippocampal neurons (FIG. 21C). The disruption of the GAPDH-GluR2
interaction by the extracellular application of the interfering
GluR2 NT 1-3-2 peptide suggested that the GAPDH and GluR2 complex
formation may occur extracellularly. Indeed, a recent study
demonstrated that in several mammalian cell lines, including
HEK-293 cells and neuro-2a cells, GAPDH was constitutively secreted
into the extracellular space (101). Furthermore, the GluR2.sub.NT
interacting proteins Narp and N-cadherins are also extracellular
proteins (24, 90). Thus, without wishing to be bound by theory, it
is possible that secreted GAPDH may form a protein complex with
GluR2.sub.NT. We first confirmed GAPDH secretion in our cell lines
by immunoprecipiting GAPDH from the conditioned medium of
hippocampal primary cultures with a primary antibody against GAPDH
(rabbit polyclonal). As shown in FIG. 21D, GAPDH was
immunoprecipitated from serum-free conditioned medium, but not from
fresh serum-free medium. To further clarify that the GAPDH from
conditioned medium was secreted from cells and not a result from
cell lysis, serum-free conditioned medium of nontransfected HEK-293
cells and cells co-expressing GluR 1/GluR2 was collected,
concentrated and examined by Western blot analyses using anti-GAPDH
and anti-.alpha.-tubulin antibodies. As shown in FIG. 21E,
regardless of GluR 1/GluR2 coexpression, GAPDH was detected from
both conditioned media and cell lysates while .alpha.-tubulin (an
intracellular protein marker) was only detected from the cell
lysates, indicating that GAPDH observed in the conditioned medium
was secreted from cells and not a contaminant from cell lysis.
Furthermore, to test whether GAPDH and GluR2 interaction occurred
extracellularly, we performed cell surface biotinylation
experiments in hippocampal neurons. As shown in FIG. 21F, GluR2
antibody co-immunoprecipitated GAPDH from the biotinylated (cell
surface) fraction, but failed to co-immunoprecipitate GAPDH from
the non-biotinylated (intracellular) fraction. These data together
strongly suggested that GAPDH is secreted to extracellular space
where it is accessible for interaction with the N-terminus of the
GluR2 subunit.
Example 9
Activation of AMPAR Induces GAPDH Internalization Through
GAPDH-GluR2 Interaction
Previous studies have demonstrated agonist induced AMPAR
endocytosis (53. Carroll et al., 1999; 76. Lin et al., 2000; 82.
Man et al., 2000). Thus, we examined whether extracellular GAPDH
would internalize along with AMPARs through the GAPDH-GluR2
interaction upon agonist stimulation of AMPAR. Consistent with our
hypothesis, glutamate stimulation (100 .mu.M, 30 min) induced a
significant decrease in not only GluR2 plasma membrane localization
(FIG. 22A) but also in cell surface-associated GAPDH (FIG. 22B) in
cells co-expressing GluR1/GluR2 as indexed by cell based ELISA
assay. The ability of the GluR2 NT1-3-2 peptide to abolish the
glutamate induced decrease in GAPDH plasma membrane expression
(FIG. 3B), together with the inability of glutamate stimulation to
internalize GAPDH in the absence of GluR1/GluR2 subunits in
HEK-293T cells (FIG. 22C), suggested that the observed GAPDH
internalization maybe a passive process enabled by the GAPDH-GluR2
interaction and dependent on GluR2 internalization. The essential
role of GluR2 subunit in the GAPDH internalization was also
confirmed in GluR1/GluR3 co-expressing cells, in which glutamate
stimulation failed to induce GAPDH internalization (FIG. 22D).
Previous studies showed that GluR2 endocytosis was
dynamin-dependent and that expression of the dominant-negative
dynamin mutant (K44E) is able to block GluR2 internalization (53,
82). Thus, after confirming the ability of the K44E dynamin mutant
to block GluR2 internalization (FIG. 22E), we examined whether the
dynamin mutant could also affect GAPDH internalization in cells
co-expressing GluR 1/GluR2 in HEK-293T cells. As shown in FIG. 22F,
the K44E dynamin mutant significantly inhibited glutamate induced
GAPDH internalization, indicating that GAPDH internalizes through a
dynamin dependent pathway.
Example 10
GAPDH and GluR2 Translocate to the Nucleus Through the GAPDH-GluR2
Interaction
Previous studies demonstrated that GAPDH initiates apoptotic cell
death by nuclear translocation following Siah1 binding (63, 64).
Therefore, we next examined if the internalized GAPDH could be
translocated to the nucleus upon agonist stimulation of AMPAR.
Surprisingly, not only GAPDH but GluR2 also exhibited a significant
increase in nuclear localization upon agonist stimulation (FIGS.
23A-C). Furthermore, the nuclear translocation of GAPDH and GluR2
could be blocked by the pre-incubation of GluR2 NT1-3-2 peptide in
HEK-293T cells expressing GluR 1/GluR2 (FIGS. 23A-C) or in
hippocampal neurons (FIGS. 23D-F). To confirm whether the observed
nuclear GAPDH and GluR2 originated from the cell surface,
hippocampal neurons were first labeled with sulfo-NHS-SS-Biotin and
then treated with GluR2 NT1-3-2 peptides before agonist
stimulation. Subsequently, all cell surface biotin was cleaved
leaving only the endocytosed proteins labeled with biotin. As shown
in FIG. 23G, Western blots from SDS-PAGE of nuclear extracts that
were streptavidin purified revealed that the levels of biotinylated
GAPDH and GluR2 were significantly increased in the nuclear extract
of hippocampal neurons upon agonist stimulation, a phenomenon that
could be blocked by pre-incubation with the GluR2 NT1-3-2 peptide.
Thus, AMPAR activation could lead to the co-internalization of
GAPDH and GluR2 mediated by the GAPDH-GluR2 coupling and resulted
in the translocation of GluR2 and GAPDH to the nucleus.
Example 11
Activation of AMPAR Facilitates Nuclear GAPDH-p53 Coupling
GAPDH nuclear localization was previously implicated in apoptosis
(25. Chuang et al., 2005) and p53, a tumor suppressor and
transcription factor, which can also initiate apoptosis, has been
implicated in glutamate-mediated excitotoxicity (72, 91, 95). More
interestingly, a previous study showed an interaction between GAPDH
and p53 (45). Thus, we tested whether GluR2.sub.NT and GAPDH can
interact with p53 using affinity "pull down" purification
experiments. Interestingly, only GST-GAPDH, but not
GST-GluR2.sub.NT or GST alone, affinity precipitated p53 from
nuclear extracts of rat hippocampal neurons (FIG. 24A). In
addition, as shown in FIG. 24B, GAPDH co-immunoprecipitated with
p53 taken from isolated nuclei of primary cultures of hippocampal
neurons, indicating a physical interaction exists between GAPDH and
p53, an interaction that appears to be facilitated by AMPAR
activation. Furthermore, we found that p53 acted as a competitive
inhibitor to GAPDH-GluR2 coupling since pretreatment with the
interfering GluR2 NT1-3-2 peptide, which we have shown to disrupt
the GAPDH-GluR2 interaction, also inhibited the GAPDH-p53
interaction (FIG. 24B), and pre-incubation with purified p53-GST,
but not GST alone, inhibited GluR2-GAPDH coupling in a
concentration dependent manner, as indexed by affinity "pull down"
experiments (FIG. 24C). To identify the p53 interacting domain on
GAPDH, GST-fusion proteins encoding truncated fragments of GAPDH
were constructed and used in affinity purification assays (FIG.
24D). These results revealed that the sequence encoded by the
GAPDH: I221-E250 facilitates the interaction with p53 since only
the GST-GAPDH(2-2-1-1) was able to pull-down p53 from solubilized
nuclear proteins extracted from rat hippocampus (FIG. 24E-H).
Furthermore, we confirmed the essential role of I221-E250 in
maintaining GAPDH-p53 coupling. As shown in FIG. 24I, co-expression
of the GAPDH(2-2-1-1) mini-gene was able to block
co-immunoprecipitation of p53 with GAPDH.
Example 12
Both GluR2-GAPDH and GAPDH-p53 Play Roles in GluR2-Containing
AMPAR-Mediated Cell Death
AMPAR endocytosis was recently shown to be required for excitotoxic
neuronal injury (Wang et al, 2004). Moreover, both GAPDH and p53
have been independently shown to be involved in cell toxicity (4,
25, 3). Therefore, we suspected that the sequential internalization
and protein-protein interactions among GluR2, GAPDH and p53 may
play an essential role in mediating AMPAR-induced excitotoxicity.
Consistent with previously studies (52, 67), treatment of HEK-293T
cells expressing GluR1/2 with glutamate (300 .mu.M, 24 hour; plus
25 .mu.M cyclothiazide to prevent AMPAR desensitization) produced
significant cell death (FIG. 25A). Given that excessive influx of
Ca.sup.2+ through glutamate receptor channels is thought to be
responsible for glutamate induced cell death, we then examined the
role of extracellular Ca.sup.2+ in the observed GluR2-containing
AMPAR-mediated cell death. HEK-293T cells expressing either
GluR1/GluR2 or NR 1/2A were exposed to glutamate in the presence or
absence of EGTA (5 mM). As shown in FIG. 25B, in the presence of
EGTA, NMDA receptor-mediated to cell death was significantly
reduced, while the GluR1/GluR2 AMPAR-mediated cell death remains
intact, indicating that cell death induced by GluR2-containing
AMPAR may not be dependent on extracellular Ca.sup.2+ influx via
the ionotropic receptor. To investigate the involvement of
GluR2-GAPDH interaction in AMPAR-mediated cell death, we
pre-treated with the GluR2 NT1-3-2 peptide (10 .mu.M, 1 hour) in
HEK-293T cells expressing GluR1/GluR2. As shown in FIG. 25C,
pre-incubation with the GluR2 NT1-3-2 peptide attenuated
AMPAR-mediated cell death by 56.+-.1.6%, suggesting that disruption
of GAPDH-GluR2 coupling may indeed rescue cells from AMPAR mediated
cell death. The GluR2 NT1-3-2 peptide itself showed no effect on
either GluR1/2 transfected cells without glutamate treatment or in
nontransfected cells regardless of glutamate treatment (FIGS. 25C
and 25D). The specificity of the GluR2 NT1-3-2 peptide was also
confirmed in cells co-expressing GluR 1/3 subunits, where
pre-incubation with the GluR2 NT1-3-2 peptide failed to inhibit
GluR1/3 AMPAR-mediated cell death (FIG. 25E). These data suggested
that the GAPDH-GluR2 interaction may play a role in
GluR2-containing AMPAR-mediated cell death.
To examine the GAPDH: AMPAR interactions in a relevant cellular
milieu, primary cultures of rat hippocampal neurons were utilized
in parallel experiments. We have previously shown in FIG. 21C that
pre-incubating hippocampal neurons with the GluR2 NT1-3-2 peptide
could disrupt the GAPDH-GluR2 interaction that was promoted by
AMPAR activation. We subsequently examined if disruption of this
interaction in hippocampal neurons could rescue cells from
AMPAR-mediated cell death. AMPAR-mediated cell death was induced by
pretreating neurons with kainic acid (KA; 100 .mu.M, 1 hour) in the
presence of NMDA receptor and Ca.sup.2+ channel antagonists (10
.mu.M MK-801 and 2 .mu.M nimodipine, respectively). As shown in
FIG. 25F, pretreatment with the GluR2 NT1-3-2 peptide significantly
inhibited AMPAR-mediated cell death. These results suggested that
the AMPAR could functionally interact with GAPDH and that
disruption of this interaction leads to a significant decrease in
AMPA-mediated cell death in neurons.
We then investigated the role of GAPDH-p53 coupling in
GluR2-containing AMPAR-mediated neurotoxicity. As shown in FIG.
25G, pre-treating HEK-293T cells expressing GluR1/GluR2 with the
p53 antagonist PFT.alpha. (10 .mu.M, 1 hour) significantly
inhibited glutamate-induced cell death, while PFT.alpha. failed to
inhibit glutamate-induced cell death in cells expressing GluR1/3
(FIG. 25H), suggesting that GluR2-containing AMPAR induces cell
death through a p53-dependent pathway. To examine whether GAPDH-p53
coupling plays a functional role in GluR2-containing AMPAR induced
cell death we co-transfected a mini-gene encoding the
GAPDH(2-2-1-1) in HEK-293T cells co-expressing GluR1/GluR2, which
results in the disruption of the GAPDH-p53 interaction as
previously shown in co-immunoprecipitation experiments (FIG. 24I).
As shown in FIG. 25I, agonist induced GluR2-containing
AMPAR-mediated cell death was significantly inhibited in cells
co-expressing the GAPDH(2-2-1-1) mini-gene, indicating the critical
role of GAPDH-p53 coupling in GluR2-containing AMPAR-mediated cell
death. Previous studies demonstrated a strong correlation between
p53 expression and excitotoxic neuronal death (72, 91, 95), while
other studies reported phosphorylation can regulate p53 activity
(51). Thus, we tested whether enhancing the GAPDH-p53 coupling by
AMPAR activation affects p53 expression and phosphorylation. As
shown in FIG. 25J, both the expression of p53 and the
phosphorylation of p53 were enhanced upon agonist stimulation in
cells expressing GluR 1/GluR2, but not in cells co-expressing
GluR1/GluR2 and the GAPDH(2-2-1-1) mini-gene. Together, these data
suggested that GluR2-mediated GAPDH nuclear translocation is
responsible for GluR2-containing AMPAR-mediated cell death, which
facilitates the interaction between GAPDH and p53 and activates
p53-dependent apoptosis pathway.
Example 13
Testing of GluR2 NT Mutants
Experiments were performed using mutants of sequences as shown in
FIG. 26. Nuclei from HEK-293T cells cotransfected with GluR 1/GluR2
were purified, solubilized and run on SDS-PAGE with subsequent
Western blot analysis. Both GAPDH and GluR2 nuclear expression was
significantly increased upon glutamate treatment (100 .mu.M, 30
min) and the nuclear translocation could not be blocked by
co-transfection of the GluR2.sub.220-238 mini-gene.
GluR2.sub.220-238 is the binding site of GluR2 and Siah1. The
intensity of protein bands were measured by Image J software and
normalized to the corresponding control samples. FIG. 26B shows a
schematic representation of GluR2 mutants. GluR2-M1 94-95
KK->AA; GluR2-M2 171-172 KK->AA; GluR2-M3 187-188 KK->AA.
(FIG. 26C shows both GAPDH and GluR2 nuclear expression was
significantly decreased in GluR2-M2 transfected HEK293T cells upon
glutamate treatment (100 .mu.M, 30 min). FIG. 26D shows GluR2-M2
inhibited glutamate-induced cell death in AMPAR transfected HEK293T
cells FIG. 26E shows GAPDH was immunoprecipitated by GluR2.sub.NT
wild type and GluR2.sub.NT mutants. FIG. 26F shows GluR2
translocated mainly on nuclear envelope, while GAPDH translocated
mainly into nucleoplasm after AMPA receptor activation. (see G-H),
CO-IP of GAPDH by GluR2 subunit (upper panel) and p53 (lower panel)
in nuclear envelope and nucleoplasm of rat hippocampal neurons.
Example 13
In-Vivo Neuroprotective Activity of Peptide GluR2 NT1-3-2 in an
Ischemia Model
In this study, a cannula (small diameter stainless steel tubes) was
implanted in the animal one week before surgery. This cannula was
used to deliver the peptide GluR2 NT1-3-2 (1 .mu.M, 0.5 .mu.l) into
hippocampus where GAPDH-GluR2 interaction is considered to occur.
On the surgery day, the four vessel occlusion ischemia model was
performed in order to induce ischemia. Animals were treated with
the peptide GluR2 NT1-3-2 either before (30 min) or after (2 hour)
the induction of ischemia to examine the neuroprotective effect of
the peptide. After a 5-day recirculation period, animals were
decapitated, the brains were removed and dissected to harvest the
hippocampus. Cresyl violet was used to stain alive neurons in
hippocampus region of each animal. Cresyl violet-stained nuclei
were observed by microscope and total number of stained nuclei in
CA 1 region was summarized and normalized to the sham-operated
group.
The results shown in FIG. 27 indicate that in-vivo treatment with
polypeptides of the present invention either before ischemia or
after ischemia increase neuronal survival. Specifically, peptide
treatment after ischemia rescued 13.2% neurons from cell death;
while peptide treatment before ischemia rescued 18.2% neurons from
cell death.
Example 14
GluR2 NT1-3-2 disrupts GluR2/GAPDH Formation in Rat Brain Following
an Ischemic Event
The four vessel occlusion ischemia model was performed on male
Sprague-Dawley rats. In brief, both vertebral arteries of the test
subjects were permanently occluded by electrocauterization and the
common carotid arteries were loosely snared with silk threads. An
ischemic event was triggered by placing aneurysm clips on the
common carotid arteries for 10 minutes. After 2 minutes of blood
flow occlusion, righting reflex and pupil dilation appeared and
laser Doppler perfusion monitor was applied directly and
non-invasively to measure the blood brain flow to confirm ischemia.
Two hours after the ischemic event, TAT-GluR2.sub.NT1-3-2 (0.5
.mu.l, 10 mM) was administered via stereotaxic hippocampal
injection. Sham animals received the same surgical preparation and
recovery paradigms, but no transient carotid occlusion.
Co-immunoprecipitation of GAPDH with GluR2 subunit from sham,
ischemia and TAT-GluR2.sub.NT1-3-2 peptide-treated rat brains
revealed an increase in the GluR2/GAPDH interaction in ischemia
rats, an effect that was inhibited by the TAT-GluR2.sub.NT1-3-2
peptide (FIGS. 28A and B). A similar change was observed in the
GAPDH/p53 interaction in nuclear extracts (FIGS. 28C and D). The
nuclear translocation of both GluR2 and GAPDH in ischemia rat
brains was also enhanced in the ischemia group and inhibited by the
application of the TAT-GluR2.sub.NT1-3-2 peptide (FIG. 28E-G).
The results provided herein suggest that the polypeptides of the
present invention can be employed in vivo, for example, without
limitation, to modulate AMPA receptor exitotoxicity in response to
a variety of insults or trauma. Further, the results of the present
invention suggest that the polypeptides of the present invention
may be employed as preventative agents, therapeutic agents, or
both.
The present invention has been described with regard to one or more
embodiments. However, it will be apparent to persons skilled in the
art that a number of variations and modifications can be made
without departing from the scope of the invention as defined in the
claims.
All citations are herein incorporated by reference.
References
1. Hollmann, M. & Heinemann, S. Cloned glutamate receptors.
Annu. Rev. Neurosci. 17, 31-108 (1994). 2. Bliss, T. V. P. &
Collingridge, G. L. A synaptic model of memory: Long-term
potentiation in the hippocampus. Nature 361, 31-39 (1993). 3.
Simon, R. P., Swan, J. H., and Meldrum, B. S. (1984). Blockade of
N-methyl-D-aspartate receptors may protect against ischemic damage
in the brain. Science 226, 850-852. 4. Choi, D. W. (1995). Calcium:
still center-stage in hypoxic-ischemic neuronal death. Trends
Neurosci. 18, 58-60. 5. Lee J M, Zipfel G J, Choi D W The changing
landscape of ischaemic brain injury mechanisms. Nature 1999 Jun.
24; 399 (6738 Suppl): A7-14. 6. Pulsinelli, W. A., Levy, D. E., and
Duffy, T. E. (1982). Regional cerebral blood flow and glucose
metabolism following transient forebrain ischemia. Ann Neurol 11,
499-502. 7. Schmidt-Kastner, R. and Freund, T. F., (1991).
Selective vulnerability of the hippocampus in brain ischemia.
Neuroscience 40, pp: 599-636. 8. Pellegrini-Giampietro, D. E.,
Zukin, R. S., Bennett, M. V., Choi, S, and Pulsinelli, W. A., 1992.
Switch in glutamate receptor subunit gene expression in CA 1
subfield of hippocampus following global ischemia in rats. Proc.
Natl. Acad. Sci. USA 89, pp. 10499-10503 9. Gill, R., and Lodge, D.
(1997). Pharmacology of AMPA antagonists and their role in
neuroprotection. Int Rev Neurobiol 40, 197-232. 10. Oguro, K.,
Oguro, N., Kojima, T., Grooms, S. Y., Calderone, A., Zheng, X.,
Bennett, M. V. L. and Zukin, R. S., 1999. Knockdown of AMPA
receptor GluR2 expression causes delayed neurodegeneration and
increases damage by sublethal ischemia in hippocampal CA1 and CA3
neurons. J. Neurosci. 19, pp. 9218-9227 11. Weiss, J. H. and Sensi,
S. L., 2000. Ca.sup.2+--Zn.sup.2+ permeable AMPA or kainate
receptors: possible key factors in selective neurodegeneration.
Trends Neurosci. 23, pp. 365-371. 12. Yin, H. Z., Sensi, S. L.,
Ogoshi, F., and Weiss, J. H. (2002). Blockade of Ca2+-permeable
AMPA/kainate channels decreases oxygen-glucose deprivation-induced
Zn2+ accumulation and neuronal loss in hippocampal pyramidal
neurons. J Neurosci 22, 1273-1279 13. Wang, J., Liu, S. H., Fu, Y.
P., Wang, J. H. and Lu, Y. M., 2003. CdkS activation induces CA1
pyramidal cell death by direct phosphorylating NMDA receptors. Nat.
Neurosci. 6, pp. 1039-1047. 14. Greengard, P., Jen, J., Nairn, A.
C. & Stevens, C. F. Enhancement of the glutamate response by
cAMP-dependent protein kinase in hippocampal neurons. Science 253,
1135-8. (1991). 15. Wang, L. Y., Dudek, E. M., Browning, M. D.
& MacDonald, J. F. Modulation of AMPA/kainate receptors in
cultured murine hippocampal neurones by protein kinase C. J Physiol
475, 431-7. (1994). 16. Yakel, J. L., Vissavajjhala, P., Derkach,
V. A., Brickey, D. A. & Soderling, T. R. Identification of a
Ca2+/calmodulin-dependent protein kinase II regulatory
phosphorylation site in non-N-methyl-D-aspartate glutamate
receptors. Proc Natl Acad Sci USA 92, 1376-80. (1995). 17.
Soderling, T. R. Structure and regulation of
calcium/calmodulin-dependent protein kinases II and IV. Biochim
Biophys Acta 1297, 131-8. (1996). 18. Barria, A., Derkach, V. &
Soderling, T. Identification of the Ca2+/calmodulin-dependent
protein kinase II regulatory phosphorylation site in the
alpha-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate-type
glutamate receptor. J Biol Chem 272, 32727-30. (1997). 19. Xia, J.,
Zhang, X., Staudinger, J. & Huganir, R. L. Clustering of AMPA
receptors by the synaptic PDZ domain-containing protein PICK1.
Neuron 22, 179-87. (1999). 20. Dong, H. et al. GRIP: a synaptic PDZ
domain-containing protein that interacts with AMPA receptors.
Nature 386, 279-84. (1997). 21. Osten, P. et al. The AMPA receptor
GluR2 C terminus can mediate a reversible, ATP-dependent
interaction with NSF and alpha- and beta-SNAPs. Neuron 21, 99-110.
(1998). 22. Daw, M. I. et al. PDZ proteins interacting with
C-terminal GluR2/3 are involved in a PKC-dependent regulation of
AMPA receptors at hippocampal synapses. Neuron 28, 873-86. (2000).
23. Allison, D. W., Gelfand, V. I., Spector, I. & Craig, A. M.
Role of actin in anchoring postsynaptic receptors in cultured
hippocampal neurons: differential attachment of NMDA versus AMPA
receptors. J Neurosci 18, 2423-36. (1998). 24. O'Brien, R. J., et
al., Synaptic clustering of AMPA receptors by the extracellular
immediate-early gene product Narp. Neuron, 1999. 23(2): p. 309-23.
25. Chuang, D. M., Hough, C., and Senatorov, V. V. (2005).
Glyceraldehyde-3-phosphate dehydrogenase, apoptosis, and
neurodegenerative diseases. Annu Rev Pharmacol Toxicol 45, 269-290.
26. Sirover, M. A. (2005). New nuclear functions of the glycolytic
protein, glyceraldehyde-3-phosphate dehydrogenase, in mammalian
cells. J Cell Biochem 95, 45-52. 27. Sawa, A., et al.,
Glyceraldehyde-3-phosphate dehydrogenase: nuclear translocation
participates in neuronal and normeuronal cell death. Proc Natl Acad
Sci USA, 1997. 94(21): p. 11669-74. 28. Ishitani, R., et al.,
Nuclear localization of overexpressed glyceraldehyde-3-phosphate
dehydrogenase in cultured cerebellar neurons undergoing apoptosis.
Mol Pharmacol, 1998. 53(4): p. 701-7. 29. Ishitani, R. and D. M.
Chuang, Glyceraldehyde-3-phosphate dehydrogenase antisense
oligodeoxynucleotides protect against cytosine
arabinonucleoside-induced apoptosis in cultured cerebellar neurons.
Proc Natl Acad Sci USA, 1996. 93(18): p. 9937-41. 30. tiara, M. R.,
et al., S-nitrosylated GAPDH initiates apoptotic cell death by
nuclear translocation following Siah1 binding. Nat Cell Biol, 2005.
7(7): p. 665-74. 31. Tsai, R. L. and H. Green, Studies on a
mammalian cell protein (P8) with affinity for DNA in vitro. J Mol
Biol, 1973. 73(3): p. 307-16. 32. Singh, R. and M. R. Green,
Sequence-specific binding of transfer RNA by
glyceraldehyde-3-phosphate dehydrogenase. Science, 1993. 259(5093):
p. 365-8. 33. Baxi, M. D. and J. K. Vishwanatha, Uracil
DNA-glycosylase/glyceraldehyde-3-phosphate dehydrogenase is an Ap4A
binding protein. Biochemistry, 1995. 34(30): p. 9700-7. 34. Nagy,
E. and W. F. Rigby, Glyceraldehyde-3-phosphate dehydrogenase
selectively binds AU-rich RNA in the NAD(+)-binding region
(Rossmann fold). J Biol Chem, 1995. 270(6): p. 2755-63. 35.
Schultz, D. E., C. C. Hardin, and S. M. Lemon, Specific interaction
of glyceraldehyde 3-phosphate dehydrogenase with the
5'-nontranslated RNA of hepatitis A virus. J Biol Chem, 1996.
271(24): p. 14134-42. 36. Tisdale, E. J.,
Glyceraldehyde-3-phosphate dehydrogenase is required for vesicular
transport in the early secretory pathway. J Biol Chem, 2001.
276(4): p. 2480-6. 37. Tisdale, E. J., Glyceraldehyde-3-phosphate
dehydrogenase is phosphorylated by protein kinase Ciota/lambda and
plays a role in microtubule dynamics in the early secretory
pathway. J Biol Chem, 2002. 277(5): p. 3334-41. 38. Tisdale, E. J.,
C. Kelly, and C. R. Artalejo, From ER to Golgi:
Glyceraldehyde-3-phosphate dehydrogenase interacts with Rab2 and
plays an essential role in endoplasmic reticulum to Golgi transport
exclusive of its glycolytic activity. J Biol Chem, 2004. 279(52):
p. 54046-52. 39. Kumagai, H. and H. Sakai, A porcine brain protein
(35 K protein) which bundles microtubules and its identification as
glyceraldehyde 3-phosphate dehydrogenase. J Biochem (Tokyo), 1983.
93(5): p. 1259-69. 40. Glaser, P. E., X. Han, and R. W. Gross,
Tubulin is the endogenous inhibitor of the glyceraldehyde
3-phosphate dehydrogenase isoform that catalyzes membrane fusion:
Implications for the coordinated regulation of glycolysis and
membrane fusion. Proc Natl Acad Sci USA, 2002. 99(22): p. 14104-9.
41. Sawa, A., et al., Glyceraldehyde-3-phosphate dehydrogenase:
nuclear translocation participates in neuronal and normeuronal cell
death. Proc Natl Acad Sci USA, 1997. 94(21): p. 11669-74. 42.
Ishitani, R., et al., Nuclear localization of overexpressed
glyceraldehyde-3-phosphate dehydrogenase in cultured cerebellar
neurons undergoing apoptosis. Mol Pharmacol, 1998. 53(4): p. 701-7.
43. Ishitani, R. and D. M. Chuang, Glyceraldehyde-3-phosphate
dehydrogenase antisense oligodeoxynucleotides protect against
cytosine arabinonucleoside-induced apoptosis in cultured cerebellar
neurons. Proc Natl Acad Sci USA, 1996. 93(18): p. 9937-41. 44.
Hara, M. R., et al., S-nitrosylated GAPDH initiates apoptotic cell
death by nuclear translocation following Siah1 binding. Nat Cell
Biol, 2005. 7(7): p. 665-74. 45. Chen, R. W., Saunders, P. A., Wei,
H., Li, Z., Seth, P., and Chuang, D. M. (1999). Involvement of
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and p53 in
neuronal apoptosis: evidence that GAPDH is upregulated by p53. J
Neurosci 19, 9654-9662. 46. Dastoor, Z., and Dreyer, J. L. (2001).
Potential role of nuclear translocation of
glyceraldehyde-3-phosphate dehydrogenase in apoptosis and oxidative
stress. J Cell Sci 114, 1643-1653. 47. Barbosa M. S. Bao, S. N.,
Andreotti, P. F., de Faria, F. P., Felipe, M. S., dos Santos
Feitosa, L., Mendes-Giannini, M. J., and Soares, C. M. (2006).
Glyceraldehyde-3-phosphate dehydrogenase of Paracoccidioides
brasiliensis is a cell surface protein involved in fungal adhesion
to extracellular matrix proteins and interaction with cells. Infect
Immun 74, 382-389. Bhattacharya 48. Bhattacharya, M., Peri, K.,
Ribeiro-da-Silva, A., Almazan, G., Shichi, H., Hou, X., Varma, D.
R., and Chemtob, S. (1999). Localization of functional
prostaglandin E2 receptors EP3 and EP4 in the nuclear envelope. The
Journal of biological chemistry 274, 15719-15724. 49. Bkaily, G.,
Choufani, S., Hassan, G., E1-Bizri, N., Jacques, D., and
D'Orleans-Juste, P. (2000). Presence of functional endothelin-1
receptors in nuclear membranes of human aortic vascular smooth
muscle cells. Journal of cardiovascular pharmacology 36, S414-417.
50. Bliss, T. V., and Collingridge, G. L. (1993). A synaptic model
of memory: long-term potentiation in the hippocampus. Nature 361,
31-39. 51. Brooks, C. L., and Gu, W. (2003). Ubiquitination,
phosphorylation and acetylation: the molecular basis for p53
regulation. Current opinion in cell biology 15, 164-171. 52.
Carriedo, S. G., Yin, H. Z., Sensi, S. L., and Weiss, J. H. (1998).
Rapid Ca2+ entry through Ca2+-permeable AMPA/Kainate channels
triggers marked intracellular Ca2+ rises and consequent oxygen
radical production. J Neurosci 18, 7727-7738. 53. Carroll, R. C.,
Beattie, E. C., Xia, H., Luscher, C., Altschuler, Y., Nicoll, R.
A., Malenka, R. C., and von Zastrow, M. (1999). Dynamin-dependent
endocytosis of ionotropic glutamate receptors. Proceedings of the
National Academy of Sciences of the United States of America 96,
14112-14117. 54. Chen, L., Chetkovich, D. M., Petralia, R. S.,
Sweeney, N. T., Kawasaki, Y., Wenthold, R. J., Bredt, D. S., and
Nicoll, R. A. (2000). Stargazin regulates synaptic targeting of
AMPA receptors by two distinct mechanisms. Nature 408, 936-943. 55.
Choi, Y. D., and Dreyfuss, G. (1984). Isolation of the
heterogeneous nuclear RNA-ribonucleoprotein complex (hnRNP): a
unique supramolecular assembly. Proceedings of the National Academy
of Sciences of the United States of America 81, 7471-7475. 56.
Dong, H., O'Brien, R. J., Fung, E. T., Lanahan, A. A., Worley, P.
F., and Huganir, R. L. (1997). GRIP: a synaptic PDZ
domain-containing protein that interacts with AMPA receptors.
Nature 386, 279-284. 57. Dong, H., Zhang, P., Song, I., Petralia,
R. S., Liao, D., and Huganir, R. L. (1999). Characterization of the
glutamate receptor-interacting proteins GRIP I and GRIP2. J
Neurosci 19, 6930-6941. 58. Doucet, J. P., and Tuana, B. S. (1991).
Identification of low molecular weight GTP-binding proteins and
their sites of interaction in subcellular fractions from skeletal
muscle. The Journal of biological chemistry 266, 17613-17620. 59.
Fuchtbauer, A., Jockusch, B. M., Leberer, E., and Pette, D. (1986).
Actin-severing activity copurifies with phosphofructokinase.
Proceedings of the National Academy of Sciences of the United
States of America 83, 9502-9506. 60. Geiger, J. R., Melcher, T.,
Koh, D. S., Sakmann, B., Seeburg, P. H., Jonas, P., and Monyer, H.
(1995). Relative abundance of subunit mRNAs determines gating and
Ca2+ permeability of AMPA receptors in principal neurons and
interneurons in rat CNS. Neuron 15, 193-204. 61. Gill, R., and
Lodge, D. (1997). Pharmacology of AMPA antagonists and their role
in neuroprotection. International review of neurobiology 40,
197-232. 62. Glaser, P. E., and Gross, R. W. (1995). Rapid
plasmenylethanolamine-selective fusion of membrane bilayers
catalyzed by an isoform of glyceraldehyde-3-phosphate
dehydrogenase: discrimination between glycolytic and fusogenic
roles of individual isoforms. Biochemistry 34, 12193-12203. 63.
Hara, M. R., Agrawal, N., Kim, S. F., Cascio, M. B., Fujimuro, M.,
Ozeki, Y., Takahashi, M., Cheah, J. H., Tankou, S. K., Hester, L.
D., et al. (2005). S-nitrosylated GAPDH initiates apoptotic cell
death by nuclear translocation following Siah1 binding. Nature cell
biology 7, 665-674. 64. Hara, M. R., Thomas, B., Cascio, M. B.,
Bae, B. I., Hester, L. D., Dawson, V. L., Dawson, T. M., Sawa, A.,
and Snyder, S. H. (2006). Neuroprotection by pharmacologic blockade
of the GAPDH death cascade. Proceedings of the National Academy of
Sciences of the United States of America 103, 3887-3889. 65.
Huitorel, P., and Pantaloni, D. (1985). Bundling of microtubules by
glyceraldehyde-3-phosphate dehydrogenase and its modulation by ATP.
Eur J Biochem 150, 265-269. 66. Humbert, J. P., Matter, N.,
Artault, J. C., Koppler, P., and Malviya, A. N. (1996). Inositol
1,4,5-trisphosphate receptor is located to the inner nuclear
membrane vindicating regulation of nuclear calcium signaling by
inositol 1,4,5-trisphosphate. Discrete distribution of inositol
phosphate receptors to inner and outer nuclear membranes. The
Journal of biological chemistry 271, 478-485. 67. lihara, K., Joo,
D. T., Henderson, J., Sattler, R., Taverna, F. A., Lourensen, S.,
Orser, B. A., Roder, J. C., and Tymianski, M. (2001). The influence
of glutamate receptor 2 expression on excitotoxicity in Glur2 null
mutant mice. J Neurosci 21, 2224-2239. 68. Ikemoto, A., Bole, D.
G., and Ueda, T. (2003). Glycolysis and glutamate accumulation into
synaptic vesicles. Role of glyceraldehyde phosphate dehydrogenase
and 3-phosphoglycerate kinase. The Journal of biological chemistry
278, 5929-5940. 69. Jonas, P., Racca, C., Sakmann, B., Seeburg, P.
H., and Monyer, H. (1994). Differences in Ca2+ permeability of
AMPA-type glutamate receptor channels in neocortical neurons caused
by differential GluR-B subunit expression. Neuron 12, 1281-1289.
70. Jong, Y. J., Kumar, V., Kingston, A. E., Romano, C., and
O'Malley, K. L. (2005). Functional metabotropic glutamate receptors
on nuclei from brain and primary cultured striatal neurons. Role of
transporters in delivering ligand. The Journal of biological
chemistry 280, 30469-30480. 71. Jong, Y. J., Schwetye, K. E., and
O'Malley, K. L. (2007). Nuclear localization of functional
metabotropic glutamate receptor mGlul in HEK293 cells and cortical
neurons: role in nuclear calcium mobilization and development.
Journal of neurochemistry 101, 458-469. 72. Lakkaraju, A.,
Dubinsky, J. M., Low, W. C., and Rahman, Y. E. (2001). Neurons are
protected from excitotoxic death by p53 antisense oligonucleotides
delivered in anionic liposomes. The Journal of biological chemistry
276, 32000-32007. 73. Lee, F. J., Xue, S., Pei, L., Vukusic, B.,
Chery, N., Wang, Y., Wang, Y. T., Niznik, H. B., Yu, X. M., and
Liu, F. (2002a). Dual regulation of NMDA receptor functions by
direct protein-protein interactions with the dopamine DI receptor.
Cell 111, 219-230. 74. Lee, S. H., Liu, L., Wang, Y. T., and Sheng,
M. (2002b). Clathrin adaptor AP2 and NSF interact with overlapping
sites of GluR2 and play distinct roles in AMPA receptor trafficking
and hippocampal LTD. Neuron 36, 661-674. 75. Li, S. Y., Ni, J. H.,
Xu, D. S., and Jia, H. T. (2003). Down-regulation of GluR2 is
associated with Ca2+-dependent protease activities in
kainate-induced apoptotic cell death in cultured [correction of
culturd] rat hippocampal neurons. Neuroscience letters 352,
105-108. 76. Lin, J. W., Ju, W., Foster, K., Lee, S. H., Ahmadian,
G., Wyszynski, M., Wang, Y. T., and Sheng, M. (2000). Distinct
molecular mechanisms and divergent endocytotic pathways of AMPA
receptor internalization. Nature neuroscience 3, 1282-1290. 77.
Lin, S. Y., Makino, K., Xia, W., Matin, A., Wen, Y., Kwong, K. Y.,
Bourguignon, L., and Hung, M. C. (2001). Nuclear localization of
EGF receptor and its potential new role as a transcription factor.
Nature cell biology 3, 802-808. 78. Liu, B., Liao, M., Mielke, J.
G., Ning, K., Chen, Y., Li, L., E1-Hayek, Y. H., Gomez, E., Zukin,
R. S., Fehlings, M. G., et al. (2006). Ischemic insults direct
glutamate receptor subunit 2-lacking AMPA receptors to synaptic
sites. J Neurosci 26, 5309-5319. 79. Liu, F., Wan, Q., Pristupa, Z.
B., Yu, X. M., Wang, Y. T., and Niznik, H. B. (2000). Direct
protein-protein coupling enables cross-talk between dopamine D5 and
gamma-aminobutyric acid A receptors. Nature 403, 274-280. 80. Liu,
S., Lau, L., Wei, J., Zhu, D., Zou, S., Sun, H. S., Fu, Y., Liu,
F., and Lu, Y. (2004). Expression of Ca(2+)-permeable AMPA receptor
channels primes cell death in transient forebrain ischemia. Neuron
43, 43-55. 81. Lu, D., Yang, H., Shaw, G., and Raizada, M. K.
(1998). Angiotensin II-induced nuclear targeting of the angiotensin
type 1 (AT1) receptor in brain neurons. Endocrinology 139, 365-375.
82. Man, H. Y., Lin, J. W., Ju, W. H., Ahmadian, G., Liu, L.,
Becker, L. E., Sheng, M., and Wang, Y.
T. (2000). Regulation of AMPA receptor-mediated synaptic
transmission by clathrin-dependent receptor internalization. Neuron
25, 649-662. 83. Melikian, H. E., and Buckley, K. M. (1999).
Membrane trafficking regulates the activity of the human dopamine
transporter. J Neurosci 19, 7699-7710. 84. Miller, F. D., Pozniak,
C. D., and Walsh, G. S. (2000). Neuronal life and death: an
essential role for the p53 family. Cell death and differentiation
7, 880-888. 85. Nelson, D., Goldstein, J. M., Boatright, K., Harty,
D. W., Cook, S. L., Hickman, P. J., Potempa, J., Travis, J., and
Mayo, J. A. (2001). pH-regulated secretion of a
glyceraldehyde-3-phosphate dehydrogenase from Streptococcus
gordonii FSS2: purification, characterization, and cloning of the
gene encoding this enzyme. J Dent Res 80, 371-377. 86. Nishimune,
A., Isaac, J. T., Molnar, E., Noel, J., Nash, S. R., Tagaya, M.,
Collingridge, G. L., Nakanishi, S., and Henley, J. M. (1998). NSF
binding to GluR2 regulates synaptic transmission. Neuron 21, 87-97.
87. Oguro, K., Oguro, N., Kojima, T., Grooms, S. Y., Calderone, A.,
Zheng, X., Bennett, M. V., and Zukin, R. S. (1999). Knockdown of
AMPA receptor GluR2 expression causes delayed neurodegeneration and
increases damage by sublethal ischemia in hippocampal CA1 and CA3
neurons. J Neurosci 19, 9218-9227. 88. Osten, P., Srivastava, S.,
Inman, G. J., Vilim, F. S., Khatri, L., Lee, L. M., States, B. A.,
Einheber, S., Milner, T. A., Hanson, P. I., et al. (1998). The AMPA
receptor GluR2 C terminus can mediate a reversible, ATP-dependent
interaction with NSF and alpha- and beta-SNAPs. Neuron 21, 99-110.
89. Robbins, A. R., Ward, R. D., and Oliver, C. (1995). A mutation
in glyceraldehyde 3-phosphate dehydrogenase alters endocytosis in
CHO cells. J Cell Biol 130, 1093-1104. 90. Saglietti, L., Dequidt,
C., Kamieniarz, K., Rousset, M. C., Valnegri, P., Thoumine, O.,
Beretta, F., Fagni, L., Choquet, D., Sala, C., et al. (2007).
Extracellular interactions between GluR2 and N-cadherin in spine
regulation. Neuron 54, 461-477. 91. Sakhi, S., Bruce, A., Sun, N.,
Tocco, G., Baudry, M., and Schreiber, S. S. (1994). p53 induction
is associated with neuronal damage in the central nervous system.
Proceedings of the National Academy of Sciences of the United
States of America 91, 7525-7529. 92. Schmidt-Kastner, R., and
Freund, T. F. (1991). Selective vulnerability of the hippocampus in
brain ischemia. Neuroscience 40, 599-636. 93. Seifert, K. N.,
McArthur, W Y., Bleiweis, A. S., and Brady, L. J. (2003).
Characterization of group B streptococcal
glyceraldehyde-3-phosphate dehydrogenase: surface localization,
enzymatic activity, and protein-protein interactions. Can J
Microbiol 49, 350-356. 94. Srivastava, S., Osten, P., Vilim, F. S.,
Khatri, L., Inman, G., States, B., Daly, C., DeSouza, S., Abagyan,
R., Valtschanoff, J. G., et al. (1998). Novel anchorage of GluR2/3
to the postsynaptic density by the AMPA receptor-binding protein
ABP. Neuron 21, 581-591. 95. Uberti, D., Belloni, M., Grilli, M.,
Spano, P., and Memo, M. (1998). Induction of tumour-suppressor
phosphoprotein p53 in the apoptosis of cultured rat cerebellar
neurones triggered by excitatory amino acids. Eur J Neurosci 10,
246-254. 96. Valtschanoff, J. G., Burette, A., Davare, M. A.,
Leonard, A. S., Hell, J. W., and Weinberg, R. J. (2000). SAP97
concentrates at the postsynaptic density in cerebral cortex. Eur J
Neurosci 12, 3605-3614. 97. Ventura, C., Maioli, M., Pintus, G.,
Posadino, A. M., and Tadolini, B. (1998). Nuclear opioid receptors
activate opioid peptide gene transcription in isolated myocardial
nuclei. The Journal of biological chemistry 273, 13383-13386. 98.
Weiss, J. H., and Sensi, S. L. (2000). Ca2+--Zn2+ permeable AMPA or
kainate receptors: possible key factors in selective
neurodegeneration. Trends in neurosciences 23, 365-371. 99.
Wyszynski, M., Valtschanoff, J. G., Naisbitt, S., Dunah, A. W.,
Kim, E., Standaert, D. G., Weinberg, R., and Sheng, M. (1999).
Association of AMPA receptors with a subset of glutamate
receptor-interacting protein in vivo. J Neurosci 19, 6528-6537.
100. Xia, J., Zhang, X., Staudinger, J., and Huganir, R. L. (1999).
Clustering of AMPA receptors by the synaptic PDZ domain-containing
protein PICK1. Neuron 22, 179-187. 101. Yamaji, R., Chatani, E.,
Harada, N., Sugimoto, K., Inui, H., and Nakano, Y. (2005).
Glyceraldehyde-3-phosphate dehydrogenase in the extracellular space
inhibits cell spreading. Biochimica et biophysica acta 1726,
261-271. 102. Yin, H. Z., Sensi, S. L., Ogoshi, F., and Weiss, J.
H. (2002). Blockade of Ca2+-permeable AMPA/kainate channels
decreases oxygen-glucose deprivation-induced Zn2+ accumulation and
neuronal loss in hippocampal pyramidal neurons. J Neurosci 22,
1273-1279. 103. Zeevalk, G. D., Schoepp, D., and Nicklas, W. J.
(1995). Excitotoxicity at both NMDA and non-NMDA glutamate
receptors is antagonized by aurintribarboxylic 10 acid: evidence
for differing mechanisms of action. Journal of neurochemistry 64,
1749-1758.
SEQUENCE LISTINGS
1
11131PRTArtificial SequenceY142-K172 of GluR2 subunit receptor from
homo sapiens 1Tyr Tyr Gln Trp Asp Lys Phe Ala Tyr Leu Tyr Asp Ser
Asp Arg Gly 1 5 10 15 Leu Ser Thr Leu Gln Ala Val Leu Asp Ser Ala
Ala Glu Lys Lys 20 25 30 230PRTArtificial SequenceI221-E250 of
GAPDH polypeptide from homo sapiens 2Ile Pro Glu Leu Asn Gly Lys
Leu Thr Gly Met Ala Phe Arg Val Pro 1 5 10 15 Thr Ala Asn Val Ser
Val Val Asp Leu Thr Cys Arg Leu Glu 20 25 30 31635DNARattus
norvegicus 3atgcaaaaga ttatgcatat ttctgtcctc ctttctcctg ttttatgggg
actgattttt 60ggtgtctctt ctaacagcat acagataggg gggctatttc caaggggcgc
tgatcaagaa 120tacagtgcat ttcgggtagg gatggttcag ttttccactt
cggagttcag actgacaccc 180catatcgaca atttggaggt agccaacagt
ttcgcagtca ccaatgcttt ctgctcccag 240ttttcaagag gagtctacgc
aatttttgga ttttatgaca agaagtctgt aaataccatc 300acatcattct
gtgggacact ccatgtgtcc ttcatcacac ctagcttccc aacagatggc
360acacatccat ttgtcatcca gatgcgacct gacctcaaag gagcactcct
tagcttgatt 420gagtactacc aatgggacaa gttcgcatac ctctatgaca
gtgacagagg cttatcaaca 480ctgcaagctg ttctggattc tgctgcagag
aagaagtggc aggtgactgc tatcaatgtg 540gggaacatca acaatgacaa
gaaagatgag acctacagat cgctctttca agatctggag 600ttaaaaaaag
aacggcgtgt aatcctggac tgtgaaaggg ataaagtaaa tgacattgtg
660gaccaggtta ttaccattgg aaaacatgtt aaagggtacc attatatcat
tgcaaatctg 720ggattcactg atggggacct gctgaaaatt cagtttggag
gagcaaatgt ctctggattt 780cagattgtag actacgatga ttccctggtg
tctaaattta tagaaagatg gtcaacactg 840gaagagaaag aataccctgg
agcacacaca gcgacaatta agtatacttc ggccctgacg 900tatgatgctg
tccaagtgat gactgaagca ttccgtaacc ttcggaagca gaggattgaa
960atatcccgga gaggaaatgc aggggattgt ttggccaacc cagctgtgcc
ctggggacaa 1020ggggtcgaaa tagaaagggc cctcaagcag gttcaagttg
aaggcctctc tggaaatata 1080aagtttgacc agaatggaaa acgaataaac
tacacaatta acatcatgga gctcaaaaca 1140aatggacccc ggaagattgg
gtactggagt gaagtggata aaatggttgt caccctaact 1200gagctcccat
caggaaatga cacgtctggg cttgaaaaca agactgtggt ggtcaccaca
1260atattggaat ctccatatgt tatgatgaag aaaaatcatg aaatgcttga
agggaatgag 1320cgttacgagg gctactgtgt tgacttagct gcagaaattg
ccaaacactg tgggttcaag 1380tacaagctga ctattgttgg ggatggcaag
tatggggcca gggatgccga caccaaaatt 1440tggaatggta tggttggaga
gcttgtctac gggaaagctg acattgcaat tgctccatta 1500actatcactc
tcgtgagaga agaggtgatt gacttctcca agcccttcat gagtcttgga
1560atctctatca tgatcaagaa gcctcagaag tccaaaccag gagtgttttc
ctttcttgat 1620cctttagcct atgag 16354524PRTRattus norvegicus 4Val
Ser Ser Asn Ser Ile Gln Ile Gly Gly Leu Phe Pro Arg Gly Ala 1 5 10
15 Asp Gln Glu Tyr Ser Ala Phe Arg Val Gly Met Val Gln Phe Ser Thr
20 25 30 Ser Glu Phe Arg Leu Thr Pro His Ile Asp Asn Leu Glu Val
Ala Asn 35 40 45 Ser Phe Ala Val Thr Asn Ala Phe Cys Ser Gln Phe
Ser Arg Gly Val 50 55 60 Tyr Ala Ile Phe Gly Phe Tyr Asp Lys Lys
Ser Val Asn Thr Ile Thr 65 70 75 80 Ser Phe Cys Gly Thr Leu His Val
Ser Phe Ile Thr Pro Ser Phe Pro 85 90 95 Thr Asp Gly Thr His Pro
Phe Val Ile Gln Met Arg Pro Asp Leu Lys 100 105 110 Gly Ala Leu Leu
Ser Leu Ile Glu Tyr Tyr Gln Trp Asp Lys Phe Ala 115 120 125 Tyr Leu
Tyr Asp Ser Asp Arg Gly Leu Ser Thr Leu Gln Ala Val Leu 130 135 140
Asp Ser Ala Ala Glu Lys Lys Trp Gln Val Thr Ala Ile Asn Val Gly 145
150 155 160 Asn Ile Asn Asn Asp Lys Lys Asp Glu Thr Tyr Arg Ser Leu
Phe Gln 165 170 175 Asp Leu Glu Leu Lys Lys Glu Arg Arg Val Ile Leu
Asp Cys Glu Arg 180 185 190 Asp Lys Val Asn Asp Ile Val Asp Gln Val
Ile Thr Ile Gly Lys His 195 200 205 Val Lys Gly Tyr His Tyr Ile Ile
Ala Asn Leu Gly Phe Thr Asp Gly 210 215 220 Asp Leu Leu Lys Ile Gln
Phe Gly Gly Ala Asn Val Ser Gly Phe Gln 225 230 235 240 Ile Val Asp
Tyr Asp Asp Ser Leu Val Ser Lys Phe Ile Glu Arg Trp 245 250 255 Ser
Thr Leu Glu Glu Lys Glu Tyr Pro Gly Ala His Thr Ala Thr Ile 260 265
270 Lys Tyr Thr Ser Ala Leu Thr Tyr Asp Ala Val Gln Val Met Thr Glu
275 280 285 Ala Phe Arg Asn Leu Arg Lys Gln Arg Ile Glu Ile Ser Arg
Arg Gly 290 295 300 Asn Ala Gly Asp Cys Leu Ala Asn Pro Ala Val Pro
Trp Gly Gln Gly 305 310 315 320 Val Glu Ile Glu Arg Ala Leu Lys Gln
Val Gln Val Glu Gly Leu Ser 325 330 335 Gly Asn Ile Lys Phe Asp Gln
Asn Gly Lys Arg Ile Asn Tyr Thr Ile 340 345 350 Asn Ile Met Glu Leu
Lys Thr Asn Gly Pro Arg Lys Ile Gly Tyr Trp 355 360 365 Ser Glu Val
Asp Lys Met Val Val Thr Leu Thr Glu Leu Pro Ser Gly 370 375 380 Asn
Asp Thr Ser Gly Leu Glu Asn Lys Thr Val Val Val Thr Thr Ile 385 390
395 400 Leu Glu Ser Pro Tyr Val Met Met Lys Lys Asn His Glu Met Leu
Glu 405 410 415 Gly Asn Glu Arg Tyr Glu Gly Tyr Cys Val Asp Leu Ala
Ala Glu Ile 420 425 430 Ala Lys His Cys Gly Phe Lys Tyr Lys Leu Thr
Ile Val Gly Asp Gly 435 440 445 Lys Tyr Gly Ala Arg Asp Ala Asp Thr
Lys Ile Trp Asn Gly Met Val 450 455 460 Gly Glu Leu Val Tyr Gly Lys
Ala Asp Ile Ala Ile Ala Pro Leu Thr 465 470 475 480 Ile Thr Leu Val
Arg Glu Glu Val Ile Asp Phe Ser Lys Pro Phe Met 485 490 495 Ser Leu
Gly Ile Ser Ile Met Ile Lys Lys Pro Gln Lys Ser Lys Pro 500 505 510
Gly Val Phe Ser Phe Leu Asp Pro Leu Ala Tyr Glu 515 520 5335PRThomo
sapiens 5Met Gly Lys Val Lys Val Gly Val Asn Gly Phe Gly Arg Ile
Gly Arg 1 5 10 15 Leu Val Thr Arg Ala Ala Phe Asn Ser Gly Lys Val
Asp Ile Val Ala 20 25 30 Ile Asn Asp Pro Phe Ile Asp Leu Asn Tyr
Met Val Tyr Met Phe Gln 35 40 45 Tyr Asp Ser Thr His Gly Lys Phe
His Gly Thr Val Lys Ala Glu Asn 50 55 60 Gly Lys Leu Val Ile Asn
Gly Asn Pro Ile Thr Ile Phe Gln Glu Arg 65 70 75 80 Asp Pro Ser Lys
Ile Lys Trp Gly Asp Ala Gly Ala Glu Tyr Val Val 85 90 95 Glu Ser
Thr Gly Val Phe Thr Thr Met Glu Lys Ala Gly Ala His Leu 100 105 110
Gln Gly Gly Ala Lys Arg Val Ile Ile Ser Ala Pro Ser Ala Asp Ala 115
120 125 Pro Met Phe Val Met Gly Val Asn His Glu Lys Tyr Asp Asn Ser
Leu 130 135 140 Lys Ile Ile Ser Asn Ala Ser Cys Thr Thr Asn Cys Leu
Ala Pro Leu 145 150 155 160 Ala Lys Val Ile His Asp Asn Phe Gly Ile
Val Glu Gly Leu Met Thr 165 170 175 Thr Val His Ala Ile Thr Ala Thr
Gln Lys Thr Val Asp Gly Pro Ser 180 185 190 Gly Lys Leu Trp Arg Asp
Gly Arg Gly Ala Leu Gln Asn Ile Ile Pro 195 200 205 Ala Ser Thr Gly
Ala Ala Lys Ala Val Gly Lys Val Ile Pro Glu Leu 210 215 220 Asn Gly
Lys Leu Thr Gly Met Ala Phe Arg Val Pro Thr Ala Asn Val 225 230 235
240 Ser Val Val Asp Leu Thr Cys Arg Leu Glu Lys Pro Ala Lys Tyr Asp
245 250 255 Asp Ile Lys Lys Val Val Lys Gln Ala Ser Glu Gly Pro Leu
Lys Gly 260 265 270 Ile Leu Gly Tyr Thr Glu His Gln Val Val Ser Ser
Asp Phe Asn Ser 275 280 285 Asp Thr His Ser Ser Thr Phe Asp Ala Gly
Ala Gly Ile Ala Leu Asn 290 295 300 Asp His Phe Val Lys Leu Ile Ser
Trp Tyr Asp Asn Glu Phe Gly Tyr 305 310 315 320 Ser Asn Arg Val Val
Asp Leu Met Ala His Met Ala Ser Lys Glu 325 330 335
620PRTArtificial SequenceAsp153 - Lys172 of the GluR2 N-terminus
6Asp Ser Asp Arg Gly Leu Ser Thr Leu Gln Ala Val Leu Asp Ser Ala 1
5 10 15 Ala Glu Lys Lys 20 720PRTArtificial SequenceFragment 1 is
Tyr142 - Leu161 of GluR2 N-terminus 7Tyr Tyr Gln Trp Asp Lys Phe
Ala Tyr Leu Tyr Asp Ser Asp Arg Gly 1 5 10 15 Leu Ser Thr Leu 20
821PRTRattus norvegicus 8Val Ile Ile Ser Ala Pro Ser Ala Asp Ala
Pro Met Phe Val Met Gly 1 5 10 15 Val Asn His Glu Lys 20
924PRTRattus norvegicus 9Val Ile His Asp Asn Phe Gly Ile Val Glu
Gly Leu Met Thr Thr Val 1 5 10 15 His Ala Ile Thr Ala Thr Gln Lys
20 1014PRTRattus norvegicus 10Val Pro Thr Pro Asn Val Ser Val Val
Asp Leu Thr Cys Arg 1 5 10 11333PRTRattus norvegicus 11Met Val Lys
Val Gly Val Asn Gly Phe Gly Arg Ile Gly Arg Leu Val 1 5 10 15 Thr
Arg Ala Ala Phe Ser Cys Asp Lys Val Asp Ile Val Ala Ile Asn 20 25
30 Asp Pro Phe Ile Asp Leu Asn Tyr Met Val Tyr Met Phe Gln Tyr Asp
35 40 45 Ser Thr His Gly Lys Phe Asn Gly Thr Val Lys Ala Glu Asn
Gly Lys 50 55 60 Leu Val Ile Asn Gly Lys Pro Ile Thr Ile Phe Gln
Glu Arg Asp Pro 65 70 75 80 Val Lys Ile Lys Trp Gly Asp Ala Gly Ala
Glu Tyr Val Val Glu Ser 85 90 95 Thr Gly Val Phe Thr Thr Met Glu
Lys Ala Gly Ala His Leu Lys Gly 100 105 110 Gly Ala Lys Arg Val Ile
Ile Ser Ala Pro Ser Ala Asp Ala Pro Met 115 120 125 Phe Val Met Gly
Val Asn His Glu Lys Tyr Asp Asn Ser Leu Lys Ile 130 135 140 Val Ser
Asn Ala Ser Cys Thr Thr Asn Cys Leu Ala Pro Leu Ala Lys 145 150 155
160 Val Ile His Asp Asn Phe Gly Ile Val Glu Gly Leu Met Thr Thr Val
165 170 175 His Ala Ile Thr Ala Thr Gln Lys Thr Val Asp Gly Pro Ser
Gly Lys 180 185 190 Leu Trp Arg Asp Gly Arg Gly Ala Ala Gln Asn Ile
Ile Pro Ala Ser 195 200 205 Thr Gly Ala Ala Lys Ala Val Gly Lys Val
Ile Pro Glu Leu Asn Gly 210 215 220 Lys Leu Thr Gly Met Ala Phe Arg
Val Pro Thr Pro Asn Val Ser Val 225 230 235 240 Val Asp Leu Thr Cys
Arg Leu Glu Lys Pro Ala Lys Tyr Asp Asp Ile 245 250 255 Lys Lys Val
Val Lys Gln Ala Ala Glu Gly Pro Leu Lys Gly Ile Leu 260 265 270 Gly
Tyr Thr Glu Asp Gln Val Val Ser Cys Asp Phe Asn Ser Asn Ser 275 280
285 His Ser Ser Thr Phe Asp Ala Gly Ala Gly Ile Ala Leu Asn Asp Asn
290 295 300 Ile Val Lys Leu Ile Ser Trp Tyr Asp Asn Glu Tyr Gly Tyr
Ser Asn 305 310 315 320 Arg Val Val Asp Leu Met Ala Tyr Met Ala Ser
Lys Glu 325 330
* * * * *